Zinc: an Immunomodulator of Innate Defense Against Pathogenic Infection

Zinc:

An Immunomodulator of Innate Defense Against Pathogenic Infection

Dissertation submitted to the

Division of Graduate Studies and Research

of the University of Cincinnati

In partial fulfillment of the requirements for the Degree of

DOCTOR OF PHILOSOPHY (Ph.D.)

In the Department of

Molecular Genetics, Biochemistry and Microbiology

of the College of Medicine

2013

KAVITHA SUBRAMANIAN VIGNESH

M.Sc, Vellore Institute of Technology, India, 2008

B.Sc, Bangalore University, India, 2006

Committee chair: Dr. GEORGE S. DEEPE, Jr., MD

ABSTRACT

Zinc (Zn), an essential element, critically regulates immune function. Aberrant Zn regulation is associated with inadequate innate and adaptive responses. Macrophages phagocytose fungi and play a critical role in regulating infection. These phagocytes can favor or restrict fungal survival depending on the cytokine milieu. Under resting conditions, macrophages harbor yeasts permissively, while exposure to proinflammtory cytokines results in macrophage activation and inhibition of fungal survival. The goal of this work is to uncover the significance of Zn homeostasis driven by cytokines in shaping macrophage resistance against a fungal pathogen.

The pro-inflammatory cytokine GM-CSF confers defensive properties on macrophages to inhibit growth of Histoplasma capsulatum both in vitro and in vivo. We investigated the mechanism by which GM-CSF arms macrophages to counter fungal attack. The cytokine triggered deprivation of labile Zn by enhancing binding to metallothioneins (MTs) and inducing Zn localization in the

Golgi apparatus, associated with elevated expression of Zn transporters ZNT4 and ZNT7 known to localize on the Golgi membrane. Zn deprivation enhanced hydrogen gated voltage channel

HV1 function, augmenting the production of reactive oxygen species (ROS) by NADPH oxidase.

This coordinated program of Zn modulation in activated macrophages specifically led to Zn limitation and increased oxidative stress abrogating survival of the intracellular fungus. GM-CSF triggered Zn sequestration in vivo as well as in human macrophages.

The cytokine interleukin 4 (IL-4) is permissive, reverses the growth inhibitory property of GM-CSF activated macrophages and alleviates host immunity against fungal infection. The concept that functional attributes of alternatively activated macrophages may be influenced by

Zn homeostasis has not been investigated. We found that IL-4 treated macrophages, in sharp contrast to GM-CSF activated macrophages, enhanced the content of labile Zn and increased Zn

ii acquisition by intracellular yeasts. IL-4, in a STAT6 dependent manner increased expression of the brain specific form of MT, Mt3, which was the key player in elevating labile Zn in macrophages. Proteomic analysis identified cathepsins in IL-4 treated macrophages. Broadly targeting cathepsins using inhibitors had an attenuating effect on labile Zn increase. Both, RNA interference of MT3 and inhibition of cathepsins reduced Zn acquisition by intracellular yeasts, suggesting their role in increasing Zn bio-availability. These observations illuminate a role for

IL-4 in Zn modulation and release, and its‟ impact on directing the outcome of host defense against infection.

In summary, using a multidisciplinary approach involving immunology, mass spectrometry and bio-informatics analysis, the investigation in this work provides novel insights into metal regulatory mechanisms in innate defense against pathogen attack.

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ACKNOWLEDGEMENTS

“This work is dedicated to my parents, Kamala Subramanian and Subramanian Srinivasan,

grandparents, and my husband Vignesh Sankaran.”

First, I am immensely grateful to my parents for their undeterred support and for placing confidence in my education throughout; without them, this effort would certainly not have been possible. I am very thankful to my elder sisters, Shobha and Jayanthi who provided excellent advice during difficult times, and have been my source of courage and great direction.

I deeply thank Dr. George S. Deepe, Jr. for his distinctive mentorship, a unique combination of unconditional guidance, enormous resourcefulness and freedom to pursue unbound scientific exploration. His mentorship has carved out in me, a great appreciation for scientific, rationalistic and unconventional thinking.

I sincerely thank Dr. Julio F. Landero, an exceptional scientist, an excellent colleague and friend, and Dr. Joseph A. Caruso and Dr. Aleksey Porollo for their extensive collaboration, which has been instrumental in viewing the immune system from a chemistry and bioinformatics perspective. I am very grateful to my committee members, Dr. Edmund Choi, Dr.

John Monaco, Dr. William Miller and Dr. David Hildeman whose valuable input, constructive suggestions and direction have greatly influenced the success of this work. I also thank the past and present lab members, and the researchers from several labs that provided resources at critical times to make this pursuit a success.

Finally, I whole-heartedly thank Capt. Vignesh Sankaran, my incredible friend, and husband for giving me the invaluable gift of kindness at every stage, from a distance, thousands of miles apart.

TABLE OF CONTENTS PAGE # PAGE

Abstract ...... ii Acknowledgements ...... v Table of contents ...... vi List of Figures and Tables...... ix Abbreviations ...... xi

CHAPTER 1: Introduction ...... 1

Metals in the immune system ...... 2 (i) Role of Zn in immune function ...... 2 (ii) Zn and development of the immune system ...... 3 (iii) Zn in innate and adaptive immune responses against infection ...... 3 Zn regulatory mechanisms ...... 5 (i) Zn transporters ...... 5 (ii) Zn binding proteins ...... 8 (iii) Zn storage ...... 10 Immune response to fungal infection ...... 11 (i) Histoplasma capsulatum ...... 11 (ii) Immune response to H. capsulatum ...... 11 (iii) GM-CSF ...... 12 (iv) IL-4 ...... 14 Nutritional immunity in growth restriction ...... 14 An interdisciplinary approach to understand immune Zn regulation ...... 16 Synopsis ...... 18

CHAPTER 2: Granulocyte Macrophage-Colony Stimulating Factor-induced Zn Sequestration Enhances Macrophage Superoxide and Limits Intracellular Pathogen Survival ...... 19

Highlights ...... 20 Graphical Abstract ...... 20 Summary ...... 22 Introduction ...... 23 Results ...... 25 Discussion ...... 35 Experimental Procedures ...... 40 Figure legends ...... 45 Figures...... 50 Supplemental Information ...... 61 Inventory ...... 61 Supplemental Figures...... 62 Proteomics Report ...... 73 Supplemental Experimental Procedures ...... 83

CHAPTER 3: IL-4 Regulates Zn Homeostasis to Weaken Macrophage Defense Against an Intracellular Pathogen ...... 90

Summary ...... 91 Introduction ...... 92 Results ...... 95 Discussion ...... 100 Experimental Procedures ...... 104 Figure legends ...... 107 Figures...... 110

CHAPTER 4: Selectivity and Specificity of Small Molecule Fluorescent Dyes/Probes used for the Detection of Zn2+ and Ca2+ in Cells ...... 116

Abstract ...... 117 Introduction ...... 118 Experimental ...... 121 Results ...... 125 Discussion ...... 137 Conclusion ...... 140 Figure Legends...... 142 Figures...... 145 Tables ...... 153 Supplemental Information ...... 155 Inventory ...... 155 Supplemental Figures...... 156 Supplemental Table ...... 157

CHAPTER 5: Discussion and Future Directions ...... 162 Discussion...... 163 Review Article - Zinc Sequestration: Arming Phagocyte Defense Against Fungal Attack

Introduction ...... 163 Zinc takes center stage: A common requisite in host-pathogen interactions ...... 163 Zn acquisition strategies: Host versus Fungi ...... 164 Host Zn pool: Restricted access ...... 167 Zn regulation: An impact beyond nutritional immunity ...... 168 Figure Legends...... 170 Figures...... 172

Future Directions ...... 174 Mechanisms of Zn regulation by IL-4 ...... 174 Zn mobilization in IL-4 stimulated macrophages ...... 174 Molecular mechanism of Zn release by IL-4 ...... 175 Specificity of MT3 action in IL-4 driven labile Zn release ...... 178 Alleviation of oxidative stress ...... 179

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Zn regulation in alternative macrophage activation ...... 179 Labile Zn increase as marker for alternative activation ...... 180 Zn regulation in lysosome function ...... 180 Inhibition of phagolysosomal fusion ...... 181 Giant cell formation ...... 182

References ...... 185

viii

LIST OF FIGURES AND TABLES PAGE #

Chapter 2:

Graphical Abstract ...... 20

Main Figures (pages 50 - 59)

Figure 1: GM-CSF distinctly modulates Zn-distribution in infected macrophages ...... 50 Figure 2: GM-CSF alters Zn transporter and MT expression ...... 51 Figure 3: GM-CSF, but not TNF-triggers Zn mobilization...... 52 Figure 4: GM-CSF engages the STAT5 and STAT3 transcriptional program to regulate MT-Zn sequestration ...... 54 Figure 5: Early regulation of Mt genes and role of Slc39a2 in MT-Zn sequestration ...... 55 Figure 6: Zn sequestration enhances ROS production ...... 57 Figure 7: GM-CSF triggers Zn binding to MTs in vivo and in human macrophages ...... 59

Supplementary Figures and Tables (pages 62 – 73)

Figure S1: Chromatographic and spectral behavior of MT-Zn signal in macrophages Related to Figure 1 ...... 62 Figure S2: GM-CSF specifically alters Zn transporter and MT regulation. Related to Figure 2 ...... 64 Figure S3: Sensitivity of Zinpyr-1 and GM-CSF driven Zn localization in alveolar macrophages. Related to Figure 3 ...... 66 Figure S4: Role of Slc39a2 and Slc39a14 in GM-CSF function Related to Figure 5 ...... 68 Figure S5: Regulation of Hvcn1 expression and its role in superoxide burst Related to Figure 6 ...... 70 Figure S6: Zn regulation by GM-CSF occurs across different strains of mice and clade of H. capsulatum. Related to Figure 7 ...... 71 Table S1: Summary of protein IDs obtained from proteomics experiments Related to Figure 1 ...... 73

Chapter 3: (pages 110 – 115)

Figure 1: IL-4 increases macrophage-labile Zn and acquisition by H. capsulatum ...... 110 Figure 2: IL-4 upregulates the expression of Mt3 and Slc30a4 via STAT6 ...... 111 Figure 3: Mt3 and Slc30a4 mediate labile Zn increase by IL-4 ...... 113 Figure 4: Cathepsins regulate MT3 mediated Zn delivery to intracellular yeasts ...... 114 Figure 5: Schematic illustration of Zn regulation by IL-4 ...... 115

Chapter 4:

Main Figures (pages 145 – 154)

Figure 1: Specificity and selectivity of Zn2+ binding dyes ...... 145 Figure 2: Zoom in of the LMM region of SEC-ICP-MS trace shown in Figure 1 ...... 146 Figure 3: Specificity and selectivity of Calcium Green-1TM AM ...... 147 Figure 4: Specificity and selectivity of Oregon Green® 488 BAPTA-1 ...... 148 Figure 5: Specificity and selectivity of Fura redTM AM ...... 149 Figure 6: Specificity and selectivity of Fluo-4 NW ...... 150 Figure 7: Specificity and selectivity of Fura redTM AM with exogenous stimulation ...... 151 Figure 8: Specificity and selectivity of Fluo-4 NW with exogenous stimulation ...... 152 Table 1: FLD conditions for each probe studied ...... 153 Table 2: Summary of selectivity results for the Zn2+ and Ca2+ binding probes ...... 154

Supplementary Figures and Tables (pages 156 – 157)

Supplementary Figure S1: Unstained BMCs (negative) control ...... 156 Supplementary Table S1: Summary of protein IDs found in the 20 min SEC-ICP-MS Fraction ...... 157

Chapter 5: (pages 171 – 172)

Figure 1: Schematic of Zn regulation in phagocytes ...... 172 Figure 2: Schematic of Zn regulation in activated macrophages infected with a fungal pathogen ...... 173

ABBREVIATIONS

AAS, Atomic absorption spectroscopy ICP-MS, Inductively coupled plasma-mass ALLM, Cathepsin inhibitor spectrometry AM, acetoxy methyl ester IFN, Interferon Asp, Aspartic acid IKK, IkB kinase BMC, Bone marrow cell line IL, Interleukin Ca, Calcium IL-4Rα, IL-4 receptor alpha cAMP, Cyclic adenosine monophosphate JAK, Janus kinase CCCP, Carbonyl cyanide m-chlorophenyl LAMP, Lysosome associated macrophage hydrazine protein CCR, Chemokine receptor LMM, Low molecular mass Cd, Cadmium LPS, Lipopolysaccharide CD, Cluster of differentiation MCPIP, Monocyte chemotactic protein CDF, Cation diffusion facilitator inducing protein CFU, Colony forming units M-CSF, Macrophage-colony stimulating CIS-1, Cytokine inducible inhibitor of factor STAT signaling-1 MFI, Mean fluorescent intensity CREB, cAMP response element binding MHC, Major histocompatibility complex protein Mn, Manganese Csf2ra, Gene for GM-CSF receptor MRE, Metal response element Cu, Copper MT, Metallothionein Cys, Cysteine MTF-1, Metal transcription factor-1 DAD, Diode array detector MWCO, Molecular weight cut off DCs, Dendritic cells NADPH, Nicotinamide adenin dinucleotide DHE, Dihydroxyethidium phosphate DMSO, Dimethyl sulfoxide Ncf1, Neutrophil cytosolic factor-1 DTT, Di-thio threitol NFAT, Nuclear factor of activated-T cells ER, Endoplasmic reticulum NFkB, Nuclear factor kappa b ESI, Electrospray ionization NK, Natural killer Fe, Iron NO, Nitric oxide GFP, Green fluorescent protein Nox, NADPH oxidase Glu, Glutamine Nramp1, Non-resistance associated GM-CSF, Granulocyte macrophage-colony macrophage protein-1 stimulating factor PBMC, Peripheral blood mononuclear cells GM-CSFR, Granulocyte macrophage- Pep A, Pepstatin A, cathepsin inhibitor colony stimulating factor PMA, Phorbol myristate acetate H. capsulatum, Histoplasma capsulatum ROS, Reactive oxygen species His, Histidine S, Sulfur HMM, High molecular mass SDS, Sodium dodecyl sulfate HPLC, High performance liquid Se, Selenium chromatography SEC, Size exclusion chromatography HV1, Hydrogen gated voltage proton SEC-FL, Size exclusion chromatography- channel 1 protein Fluorescence signal Hvcn1, Hydrogen gated voltage proton siRNA, Small interfering RNA channel 1 gene Slc30a, Solute carrier family 30, member a

Slc39a, Solute carrier family 39, member a SOD, Superoxide dismutase -S-S-, Disulfide bond STAT, Signal transducer and activator of transcription factor TLR, Toll like receptor TNF, Tumor necrosis factor TPEN, N,N,N‟,N‟-tetrakis-(2- pyridylmethyl)-ethylenediamine, Zn chelator Trp, Tryptophan Tyr, Tyrosine XO, Xanthine oxidase ZAP, Zn responsive activator protein ZIP, Zn importer Zn, Zinc ZNT, Zn exporter

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CHAPTER 1

Introduction

Metals in the immune system

Transition metals are essential for survival of all forms of life. A wide range of basic cellular functions such as enzymatic reactions, transcription and signaling depend on utilization of metals as a co-factor. In the context of immunity, metals play an integral role in development and function of the immune system. They regulate a variety of processes ranging from lymphoid organ formation to mounting of an adequate response during infection. Thus, strict metal homeostasis and regulation form critical aspects of an intact immune system.

Several metals such as Zn, copper (Cu), calcium (Ca), iron (Fe), manganese (Mn) and selenium (Se) have been investigated for their role in the immune system1, 2. A deficiency or an excess caused by dysregulation of metal homeostasis often results in dramatic alterations in the development and function of innate and adaptive responses. For example, Cu deficiency is associated with retarded lymphoid organ development, decreased anti-microbial defenses in macrophages, reduced trafficking of neutrophils to the site of infection, and lower lymphocyte numbers3. Dietary Se has been shown to boost immune function and improve CD4+ T cell recovery in HIV infection4. The deficiency of Fe impairs natural killer (NK) cell activity, reduces

T cell numbers and IL-2 production by lymphocytes5. In addition, an excess of these metals leads to cellular toxicity, thereby adversely affecting integrity of the immune system6. Thus, metal regulation is a critical aspect in development and function of the immune system.

(i) Role of Zn in immune function

Zn is the second most abundant metal in biological systems, next to Fe. The involvement of Zn as a cofactor in thousands of Zn related enzymes and proteins has gained appreciation, justifying the paramount importance of this metal in cellular and biological processes. Amongst metal cations, Zn is redox inert, which is why several enzymes, transcription factors and other proteins

2 depend on this metal for function. For decades, Zn deficiency has been the focus of poor immune defense, abnormal B and T cell function and thymic atrophy7. This section discusses the significance of the myriad roles of Zn in immune function and regulation.

(ii) Zn and development of the immune system

Zn homeostasis is essential for the development of lymphoid organs, including the thymus, spleen and lymph nodes. In the thymus, thymic epithelial cells produce a Zn-dependent nano- peptide hormone termed thymulin, which induces Thy1 expression in developing thymocytes.

Thymic involution has been strongly correlated to Zn deficiency and ageing. This is chiefly because of a reduction in Zn-bound thymulin levels in aged or Zn deficient individuals, though the production of the hormone itself is not altered. Zn supplementation completely reverses thymic involution, demonstrating the requirement of Zn for function of this hormone8. Mice fed a Zn deficient diet accumulate immature IgM expressing B cells in spleens, reduced splenocyte and lymphocyte numbers9. In contrast, altered myelopoiesis resulting in greater granulocyte and monocyte numbers may be observed. Zn may impede myelopoiesis and monocyte differentiation by negatively regulating cAMP production by adenylate cyclase10. How Zn concentration in circulation impacts specific lineages in developed lymphoid organs and lymph-node architecture has not been thoroughly investigated. Thus, Zn regulation forms an essential component of immune system development.

(iii) Zn in innate and adaptive immune responses against infection

The influence of Zn in regulating the outcome of an immune response is complex. Zn may have differential effects depending on the cell type, intracellular distribution and concentration in the

3 infected tissue. The amount of Zn in circulation, in individual cells, its occurrence in labile (free / readily exchangeable) or bound forms collectively affects cellular responses in combating infection. As exemplified above, though Zn deficiency may prove beneficial to myelopoiesis, it adversely affects the adaptive arm of immune system7. This creates an imbalance, thereby dysregulating the cooperative action of innate and adaptive immunity in resolving infection.

Therefore, the term „Zn status‟ refers to regulation of Zn homeostasis not only within cells, but the whole organism.

In general, deficiency of Zn is associated with increased susceptibility to bacterial and fungal infections. In the innate compartment, Zn regulates phagocytosis by macrophages, cytokine production, and chemotaxis by neutrophils likely by influencing microtubule assembly11. The metal triggers activation of microglia in the brain by enhancing nuclear factor- kB (NFkB) activity, increasing F4/80 surface expression and production of nitric oxide (NO)12.

In contrast, in dendritic cells (DCs), maturation upon exposure to lipopolysaccharide (LPS) is associated with a decrease in intracellular Zn. Depletion of the metal enhances major histocompatibility complex – II (MHC-II) and CD86 molecules on the surface of DCs13. These studies suggest that the impact of Zn modulation is complex, and may vary depending on the cell type.

Zn also regulates the development and function of adaptive immune responses. The regulatory role of Zn on B cell proliferation and antibody responses has been poorly investigated.

A deficiency of Zn compromises the ability to mount an adequate CD4+ T cell response due to decreased proliferation resulting from reduced IL-2 production. An imbalance in the ratio of

CD45RA+ to CD45RO+ cells caused by Zn deficiency suggests that this metal plays an important role in regulating T cell memory. Interestingly, all the above defects are reversible by Zn

4 supplementation14. More recently, a role for Zn as a signaling molecule has been described.

Rapid influx of Zn into T cells sustains T cell activation15 and induces proliferation by enhancing

IL-2 generation16. These observations suggest an important role for Zn in shaping T cell immunity.

Zn regulatory mechanisms

The total cellular Zn level is in the micromolar range, a majority of which is bound to proteins, constituting over 10% of the mammalian proteome. These proteins regulate a variety of cellular functions including mitosis, transcription, protein folding and apoptosis amongst others. The physiological labile Zn concentration is low, in the picomolar range and regulates signaling, apoptosis and Zn acquisition by newly synthesized proteins17. Such a wide association of Zn with cellular functions requires strict regulation of Zn homeostasis in the cell.

(i) Zn transporters

The Zn transporter family

In mammalian cells, the Cation Diffusion Facilitator family (CDF) includes a sub-family of Zn transporters. The two sets of Zn transporters are: Slc39a-Zn importers (ZIPs) and Slc30a-Zn exporters (ZNTs). The mammalian genome encodes 14 ZIPs and 10 ZNTs that are spatially distributed on plasma membrane, membranes of intracellular organelles and vesicles17. Such a large number of transporters programmed to mobilize Zn reinforces that the distribution of this metal is strictly regulated. Import and export of Zn is defined with respect to the cytoplasm, in that, Zn importers mediate flux of Zn from extracellular milieu and intracellular organelles into the cytoplasm, while the Zn exporters flux the metal away from the cytoplasm into organelles and extracellular environment. Transport is mediated by 6 trans-membrane domains in ZNTs and

8 transmembrane domains in ZIPs containing conserved histidine (His) rich regions and aspartic acid (Asp) residues which bind Zn and facilitate transport. His rich loops in trans-membrane proteins are a characteristic feature of Zn transporters and the number of His residues in in the loop influences transport efficiency. Zn transporters may form homo/hetero dimers to facilitate

Zn flux18. Several of these importers are upregulated during Zn deficiency, while the expression of exporters confers metal resistance to cells. Though ZNTs are likely selective transporters of

Zn alone, ZIPs may be involved in transporting Fe, Mn and Cadmium (Cd)17, 19. Of note, absence or mutations of ZIPs and ZNTs is often detrimental to survival in mouse models or results in dramatic phenotypes associated with Zn deficiency20.

Spatial Distribution

Spatial location of Zn transporters directs mobilization of Zn within the cell and extracellular environment. The ZIPs and ZNTs display a non-homogenous, tissue and organelle specific distribution in the body. These facts imply functional specificity in distributing Zn to different organelles and non-redundant function of these transporters. ZIPs 1- 6 and ZIPs 8, 10 and 14 import Zn into the cytoplasm from extracellular milieu. ZIPs 7, 8, 9 and 13 may import Zn into the cytoplasm from the Golgi and intracellular vacuoles17. Amongst exporters, ZNT1 is located on the plasma membrane, is highly responsive to toxic metal exposure and confers metal- resistance. ZNT4, 5, 6 and ZNT7 mediate cytosolic Zn efflux into the Golgi18, 21, 22. ZNT4 may also translocate Zn into the endosomal compartment23. The subcellular localization of Zn transporters may be sensitive to the cellular micro-environment.

Roles in immune function

Roles of Zn transporters in immune function have been appreciated by four studies. First, activation of T cells with DCs results in rapid Zn influx in T cells. These T cells manifest

6 enhanced Slc39a6 expression and silencing it abrogates activation induced Zn-influx15. It seems likely that early Zn influx in this system was induced by already existing transporters on the plasma membrane, given the immediate Zn response to the activation signal. Second, in sharp contrast to this, lipopolysaccharide (LPS) driven Toll like receptor 4 (TLR4) signaling leads to rapid loss of labile Zn from DCs concomitant with enhanced Slc30a1 and Slc30a4 levels and down-regulation of Slc39a6 and Slc39a10 expression. These events result in DC maturation marked by increased MHC-II and CD86 surface expression13. In linking the two studies above, one may derive that rapid loss of Zn from DCs and gain of Zn by T cells are consecutively regulated by increased Zn export from DCs and import by the latter. Third, ZIP8 may regulate

IFN- expression in human T cells by increasing lysosomal Zn import into the cytoplasm, leading to sustained phosphorylation of the transcription factor, cAMP-response element binding protein (CREB)24. Contrary to its‟ effect on microglia, Zn may have an inhibitory effect on

NFkB activation in primary monocytes and macrophages. The expression of Slc39a8 is directly controlled by NFkB. Zn import caused by ZIP8 in turn, inhibits NFkB signaling by acting on IkB kinase (IKK), thereby imposing a negative feedback mechanism25. Regulation of these transporters in phagocytes and T cells in the context of infection has been sparingly investigated.

In general, there has been a disregard for an unbiased approach in examining the dynamics of transporter expression, which may undergo spatiotemporal variation. There is also a lack of appreciation of labile Zn alteration in cells, which may result from either excessive Zn sequestration or flux across membranes. These details are critical in determining the cellular Zn status. Nonetheless, the above stated advances have provided valuable information on Zn regulation by transporters.

(ii) Zn binding proteins

The mammalian proteome possesses numerous Zn binding proteins. While some proteins require

Zn for their function, others have evolved to maintain Zn equilibrium, regulate its‟ availability for cellular processes and to alleviate toxicity caused by Zn overload. With emerging interest in metal biology of the immune system, novel roles for these proteins are being described in host defense.

Metallothioneins (MTs)

MTs are cysteine-rich Zn binding proteins that establish metal-protein interaction via a „thiol‟ group and are the primary physiological regulators of labile Zn within cells. The mouse genome encodes four Mts (Mt1-Mt4), while 16 Mt genes have been identified in humans. Mt1 and Mt2 are ubiquitous in expression, but Mt3 and Mt4 show a brain and squamous epithelium specific expression pattern respectively. MTs primarily bind Zn, but can also sequester other metals such as Cu, Se, Ni and Cd, thereby conferring heavy metal detoxification/tolerance properties to cells26.

Metal binding properties of MTs

MTs are small proteins with about 60 amino acid residues, 20 of which are highly conserved cysteines. They bind 7 Zn ions with high affinity in the picomolar range via cysteines where the sulfur atom coordinates with Zn in two metal clusters containing 3 and 4 Zn residues. In protein systems, amino acids release catalytically bound Zn in the following order His > glutamine (Glu)

> Asp > cysteine (Cys), with His residues being the most capable of releasing Zn, and Cys residues binding Zn with the highest affinity. The spectral property of MTs is distinct, in that, they lack aromatic amino acid residues26, and therefore show minimal absorption at 280 nm and maximum at 260 nm, unlike many other proteins. Therefore the signal at 260 nm serves as an

8 indicator of either the oxidation state of MTs or interaction with other MTs and/or other thiol- group containing proteins. MTs may oligomerize with the same or other members in the family via -S-S- interaction, allowing them to function as a „sink for zinc‟ within the cellular milieu27.

Although MTs act as a reservoir for Zn, this metal is redox-inert. Therefore release of Zn from

MTs must involve inter-molecular ligand interactions and oxidation of the sulfur atom in Cys residues. Scavenging of superoxides by MTs involves the aforementioned process, accompanied by Zn release27. Interaction of MTs with glutathione, ATP and GTP causes the protein to release the metal28.

Regulators of MT expression

The promoters of MT genes possess „metal responsive elements‟ (MREs) and are recognized by metal responsive element binding transcription factor -1 (MTF-1). The protein is highly sensitive to cellular Zn status, in that, an increase in Zn induces Mt expression via MTF-1 and vice versa.

Zn transporter gene family members also possess MRE sites in their promoter regions29.

However, there appear to be alternative mechanisms for induction as Mt promoters have consensus STAT binding sequences, suggesting that activation of STAT signaling may potentially trigger MT expression30.

MTs in immune response

MTs are induced under a wide range of conditions including oxidative stress and LPS stimulation. Interleukin 6 (IL-6) induces Mt expression in hepatocytes30; other cytokines such as interferon-α (IFN-α and tumor necrosis factor- (TNF- modulate MTs in a variety of systems ranging from auto-immune diseases to systemic lethal inflammatory response31, 32. However, there has been a significant lack of understanding in how these signals influence oxidation, oligomerization and Zn binding properties of MTs. This information is important, as the mere

9 presence of MTs in the cell provides little information about their co-ordination with Zn, degree of oxidation, or Zn exchange. As evident from the previous section, a strong oxidative environment, such as reactive oxygen species (ROS) or high NO may mediate Zn release from

MTs, and high Zn levels in turn triggers Mt expression.

Other Zn binding proteins

Calprotectin (S-100 Ca2+) is a calcium binding protein produced by neutrophils33. This protein sequesters Zn with nanomolar affinity by binding to histidine residues34. Members of the CCCH- zinc finger protein family, monocyte chemotactic protein induced protein (MCPIP) 1-4 encoded by four genes Zc3h12a-d negatively influence macrophage activation by LPS, TNF- induction and NO production35.

(iii) Zn storage

In mammalian cells, the net Zn concentration may range in micromolars, but the free or „labile‟

Zn content is limited to a picomolar range. It has been proposed that cells possess a reservoir of labile Zn contained within zincosomes. Cells may store excess labile Zn in these bodies and release it under deficient conditions36. In addition to Zn transporters and MTs, zincosomes represent another Zn regulatory mechanism to mediate flux.

The Golgi apparatus is an additional „zinc reservoir‟ in cells. Zn exporters, ZNT4 and

ZNT7 and importers ZIP7 and ZIP13 localize on the Golgi membrane37-39, suggesting that active

Zn transport takes place across the Golgi milieu. Using Zn responsive activator protein (ZAP) as a Zn sensor, it has been demonstrated that exposure of cells to excess Zn leads to Zn sequestration within the Golgi40. The presence of labile Zn in the Golgi may be necessitated by

10 proteins acquiring Zn in this organelle. Zn may also localize in the mitochondria and endoplasmic reticulum (ER)17, 40.

Immune response to fungal infection

(i) Histoplasma capsulatum

Fungi are a common cause of infections in the immunocompromised patient population with cancer, HIV infections or those under immunosuppressive drugs. In this work, we focused on the fungal pathogen, H. capsulatum that is highly prevalent in midwestern and southeastern regions of the United States. The strain variants of Histoplasma are H. capsulatum, H duboisii and H. farciminosum of which, H. capsulatum is the most prevalent in North America and therefore extensively studied41. In United States alone, millions of individuals have encountered H. capsulatum infection; a million new infections occur globally each year; in the endemic region of

Ohio over 90% of the population has been exposed to the fungus. The organism predominantly resides in soil and bat guano in micro-conidial and mycelial form41. An intact immune response results in asymptomatic resolution of infection, however immunocompromised patients often present life threatening disseminated infection.

(ii) Immune response to H. capsulatum

H. capsulatum gains entry into the pulmonary system via inhalation of spores, which convert to yeasts at 37 °C within the host. Upon phagocytosis by macrophages via surface receptors

CD11/18 in alveolar spaces, H. capsulatum replicates to establish a niche prior to activation of the immune system42. Thus, macrophages that have not been activated (resting) by immune mediators such as cytokines permit successful establishment of infection by the fungus. The

11 strategies employed by H. capsulatum for survival within macrophages include modulation of phagolysosomal fusion, manipulation of the acidic environment in phagosomes, and iron acquisition from ferritin43, 44. However, activation of macrophages with proinflammatory cytokines such as GM-CSF effectively curtails pathogen growth45. DCs efficiently kill the engulfed pathogen and subsequently activate T cell immunity. Eventually the fungus is encompassed within highly organized „granuloma‟ structures that curtail pathogen invasiveness, but allow survival of a small number of yeasts. The yeasts may reactivate and establish disseminated infection upon disruption of integrated host immunity46. In this regard, H. capsulatum infection resembles Mycobacterium tuberculosis (Mtb), in that, both the organisms gain residence within macrophages, establish latency, and can reactivate under immunocompromised conditions.

IFN- activates murine macrophages to inhibit intracellular growth of yeasts, likely by generation of NO from arginine. However, in human peripheral blood derived macrophagesH. capsulatum growth is not inhibited by IFN-, but by granulocyte macrophage-colony stimulating factor (GM-CSF), IL-3 and macrophage-colony stimulating factor (M-CSF)47. In contrast, alternative activation of macrophages with cytokines such as IL-4 increases permissiveness and enhances pathogen survival45, 48.

The clearance of H. capsulatum infection is driven by innate and a predominant Th1 response. The regulation of Zn by immune responses and how that influences the course of H. capsulatum is not understood. It can be predicted that a state of Zn deficiency may result in increased monocyte differentiation, but impaired phagocytosis and leukocyte recruitment.

Additionally, Zn is essential for production of Th1 response cytokines, IL-2 and IFN-γ, suggesting that, a deficiency of the metal would culminate in suboptimal Th1 responses during

12 infection. Thus, Zn regulation can impact both innate and adaptive immunity against H. capsulatum.

(iii) GM-CSF

GM-CSF is a pleiotropic glycoprotein cytokine produced by a variety of cells including macrophages, T cells, mast cells, endothelial, epithelial cells and fibroblasts. The production of

GM-CSF is under the influence of nuclear factor of activated T cells (NFAT) and it stimulates differentiation of hematopoietic stem cells into monocytes, macrophages, neutrophils and other granulocytes49. However, mice deficient in this cytokine show normal hematopoietic development, suggesting that other immune mediators may contribute to this function.

GM-CSF receptor and signaling

The cytokines GM-CSF, IL-3 and IL-5 bind high-affinity receptors composed of distinct  subunits, but a common subunit. Binding of GM-CSF to its receptor GM-CSFR, activates the

Janus Kinase 2 (JAK2) -STAT5 pathway50. GM-CSF regulates a negative feedback system via cytokine inducible inhibitor of STAT signaling-1 (CIS-1) to monitor adequate signaling and to prevent excessive inflammation51. Neutralization of GM-CSF is a potent therapy in inflammatory diseases such as multiple sclerosis and arthritis52.

GM-CSF and infection

GM-CSF knockout mice show decreased recruitment of inflammatory cells and develop alveolar proteinosis due to surfactant accumulation in the lung. Though this cytokine is dispensable for normal immune homeostasis, its absence is associated with dampened humoral and cell mediated immunity. Neutralization of GM-CSF significantly elevates fungal burden in the lungs and leads to mortality. Elevation of fungal load is a consequence of decreased IFN- and TNF- and an

13 increment in IL-4 and IL-10 levels in the lung53. This suggests that GM-CSF may regulate a balance in T cell subsets during infection.

In patients exposed to chemotherapy, administration of recombinant GM-CSF is approved by FDA for its‟ beneficial role in hematopoiesis. Since immunosuppression is often worsened by secondary fungal infections, GM-CSF is likely to dually facilitate production of white blood cells and strengthen immunity against fungi. Although the significance of this cytokine in immune activation is evident, surprisingly, the genes targeted by GM-CSF and its mechanism of function remain largely elusive. It is therefore compelling to obtain insights into the mechanism of GM-CSF during infection.

(iv) IL-4

IL-4, in contrast to GM-CSF, produces an unfavorable shift in the robustness of host resistance against fungal infection. The cytokine produced by eosinophils, basophils, mast cells and Th2 cells binds to IL-4Rα – γc complex and signals via STAT654. IL-4 polarizes macrophages to the

„alternatively activated‟ or M2 phenotype characterized by increased expression of markers such as CD206 (mannose receptor) that increases endocytosis of mannosylated ligands, CD209 (DC-

SIGN) that facilitates recognition of carbohydrate ligands on pathogens and arginase-1 that alleviates NO production55. In H. capsulatum, Cryptococcus neoformans, Candida albicans and other models of fungal infection, IL-4 weakens both innate and T cell immunity, enhancing permissiveness for pathogen survival48, 56, 57. Current understanding suggests that the increase in glucose levels, amines, Fe availability and reduced NO in IL-4 treated macrophages impair their growth inhibitory properties against intracellular pathogens55. IL-4 skews T cell differentiation to the Th2 phenotype. This leads to an immune response with enhanced IL-4 secretion, but reduced

IL-12, IFN- and GM-CSF production. In murine histoplasmosis, overexpression of IL-4 is associated with a significant increase in fungal burden and delayed clearance. The chemokine receptor CCR2 negatively regulates IL-4 production, as macrophages and DCs from mice lacking CCR2 produce exaggerated levels of this cytokine48. Thus, the role for IL-4 in infectious immunity has been vastly studied, however metal modulation by the cytokine and its impact on infectious immunity have not been investigated.

Nutritional immunity in growth restriction

From a pathogen perspective, access to nutrients is a fundamental requirement for survival and successful establishment of infection. Metals form an indispensible component of biological functions in host-pathogen interactions. „Nutriprive mechanisms‟ imply depriving the availability of essential nutrients to pathogens invading the host immune system, thereby exerting an antimicrobial effect1. The mechanisms employed by phagocytes to impose metal paucity as a means of curtailing microbial pathogenesis has recently attracted attention.

Transporters as well as binding proteins contribute to metal deprivation during infection.

IFN- acts on macrophages to alter Fe levels by reducing transferrin receptors58 and increasing the expression of iron transporter ferroportin-1. The transporter, non-resistance associated macrophage protein-1 (Nramp-1) or solute carrier family 11, member a1 (Slc11a1) transports Fe from the phagosome into the cytoplasm, thereby restricting Fe to M. avium and Leishmania major within phagosomes59, 60. Phagocytes produce siderophores such as ferritin to compete with microbes to capture Fe to limit its availability61. Calprotectin is another metal binding protein that exerts fungistatic properties by Zn restriction33. GM-CSF causes Zn deprivation in H. capsulatum phagocytosed within macrophages45. Calprotecin expression is not dramatically up

15 regulated in GM-CSF activated macrophages. Thus, the mechanism by which GM-CSF imposes

Zn restriction on the pathogen has remained unclear. On the contrary, exposure of macrophages to IL-4 modulates metal homeostasis, potentially favoring metal availability for survival of pathogens. Serum Fe levels influence secretion of IL-462, and the cytokine in turn increases transferrin expression in macrophages, suggesting improved intake and storage of Fe48. IL-4 may also increase Zn availability to intracellular fungi. H. capsulatum yeasts derived from IL-4 treated macrophages amass a higher concentration of Zn, compared to GM-CSF treated macrophages45. The mechanism by which alternative activation shapes Zn mobilization leading to enhanced acquisition by the fungus is not understood. Collectively, the innate immune system modulates Zn in response to cytokine signals, effectively shaping host defensiveness during infection.

An interdisciplinary approach to understand immune Zn regulation

Zn occurs in different forms in biological systems and is constantly redistributed in the cell in response to various stimuli. Zn may occur in labile form loosely bound to small peptides, bound to proteins via high-affinity interactions or stored in „depots‟ such as intracellular organelles.

This dynamic and complex nature of Zn localization demands a multidisciplinary approach to understand regulation at the molecular level.

Atomic absorption spectrometry (AAS), inductively coupled plasma atomic emission spectroscopy (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS) are some of the commonly used techniques for detection of elements in solutions and biological systems63.

While the former techniques have proven extremely useful in the detection of some elements, their applicability is influenced by detection limits, sample volume, time required for analysis and spectral interferences. Several elements occur in trace quantities in biological systems and

16 using primary cells derived from mice or studying mouse models in vivo pose a limitation to the amount of sample generated for analysis. Therefore, techniques that provide very low limits of detection (sub-parts per billion or ng/ml), minimal background signals from spectral interference, require smaller sample volumes and shorter throughput time for detecting multiple elements from the same sample are ideal for accurate analysis of metals in immunological systems.

Inductively coupled plasma mass spectrometry (ICP-MS) measures elements by ionizing the sample with a plasma source. ICP associated with mass spectrometer permits direct detection of the singly charged elemental ions produced in the plasma, based on their mass to charge ratio, where m/z = molecular mass. ICP-MS holds the advantage of performing multi-elemental analysis in a short time span with high reproducibility, low detection limits (parts per trillion), broad range of linear response and can be coupled to chromatographic techniques to enhance productivity in cellular analysis64, 65. To dissect macrophage Zn regulation, we coupled size exclusion chromatography (SEC) to ICP-MS to produce an SEC-ICP-MS analysis system. This combination enables analysis of Zn and other metals bound to biomolecules based on their hydrodynamic radii.

SEC-ICP-MS further coupled to proteomics generates a powerful combination of chemical and protein based analysis66 to understand regulation of Zn binding to biomolecules in response to immunological stimuli. We therefore utilized electrospray ionization (ESI)-MS-MS which provides the advantage of soft ionization of macromolecules to analyze Zn-bound proteins separated by SEC.

Visualization of metals using chemical probes is a widely used approach to analyze Zn mobilization in living cells. Some examples of Zn probes include Zinpyr-1, FluoZinTM-3 AM,

Newport GreenTM DCF and Zinquin ethyl ester. Depending on their chemistry, these probes can

17 sense protein bound Zn and/or labile Zn pool in cells yielding useful information about cellular

Zn status. Zn recognition and binding causes several fold increase in fluorescence of the chemical probe. The intensity of increase in fluorescence in the presence of a stimulus is a semi- quantitative measurement of changes in Zn concentration in the cell. While some dyes are readily permeable, others have been modified with an acetoxy methyl (AM) ester group; upon entry, cellular esterases cleave the AM group causing intracellular retention of the dye67. Though these probes exhibit Zn sensitive fluorescence and are useful indicators of cellular Zn status, information generated from their usage must be complemented with additional approaches. This is because, dye fluorescence is influenced by pH, cleavage of the AM group is vesicles, and propensity to localize in hydrophobic regions. An important issue that needs to be addressed with the use of metal binding dyes is their capability to discern labile metal from those bound to biomolecules and specificity of the dye for the metal under consideration.

Synopsis

Zn regulation is essential for adequate functioning of biological processes. The mammalian system utilizes a variety of transport, storage and mobilization systems to maintain homeostasis.

It has become increasingly evident that Zn influences important aspects of immunity, and its regulation by immune mediators can dramatically alter the hosts‟ response to an infectious process. However, an understanding of immunological mechanisms underlying Zn regulation and its impact on host defense is lacking.

In Chapter 2, we investigate the GM-CSF signaling cascade uncovering novel pathways that culminate in Zn restriction and tactfully boost macrophage defense against fungal infection.

Chapter 3 examines a contrasting, yet interesting aspect of Zn modulation in favoring pathogen

18 survival over host immunity, in a program directed by IL-4 in macrophages. With increasing interest in metal regulatory functions, visualization of metal ions using fluorescent dyes is gaining popularity in immunological investigations. Chapter 4 reveals important properties of the use of Zn2+ and Ca2+ binding dyes with respect to their selectivity for the metal in question and specificity for labile vs. bound forms. Taken together, these studies uncover novel metal- modulatory functions and strengthen the foundation for strategic „metal handling‟ in innate defense against microbial attack.

CHAPTER 2

Granulocyte Macrophage-Colony Stimulating Factor-induced Zn Sequestration Enhances

Macrophage Superoxide and Limits Intracellular Pathogen Survival£

Kavitha Subramanian Vignesh1,2†, Julio A. Landero Figueroa3†, Aleksey Porollo4, Joseph

A. Caruso3 and George S. Deepe, Jr2,5*

1Department of Molecular Genetics, Biochemistry, Microbiology and Immunology, University of Cincinnati, OH 45267 USA

2Division of Infectious Diseases, College of Medicine, University of Cincinnati, Cincinnati, OH 45267 USA.

3University of Cincinnati / Agilent Technologies Metallomics Center of the Americas, Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221 USA

4Department of Environmental Health, University of Cincinnati, Cincinnati, OH 45267 USA

5Veterans Affairs Hospital, Cincinnati, OH 45220 USA.

Contact: George S. Deepe, Jr. MD, email- [email protected], Phone-513-558-4706, Fax- 513-558-2089

†These authors contributed equally to this work.

Running Title: GM-CSF drives Zinc Sequestration

£Published in Immunity, Cell Press. Immunity 39, 1–14, October 17, 2013

HIGHLIGHTS

 Metallothioneins, or Zn-sequestering proteins, drive GM-CSF effector function

 Zn sequestration augments production of reactive oxygen species via HV1

 GM-CSF triggers Zn deprivation and enhances ROS to kill fungi in macrophages

 GM-CSF drives Zn sequestration in vivo and in human macrophages

GRAPHICAL ABSTRACT

Schematic of fungal growth arrest in macrophages by GM-CSF activation

(1) Upon encountering H. capsulatum, yeasts are engulfed in a phagosome, which fuses with a lysosome to form a phagolysosome; (2) GM-CSF binds to its‟ receptor and activates a signaling cascade engaging Jak2-STAT5 and Jak2-STAT3; (3) STAT5 and STAT3 induce transcriptional activation of MTs; (4) GM-CSF signaling increases the expression of Mt genes, Slc39a2,

Slc30a4, and Slc30a7 expression; (5) ZIP2 causes Zn influx into the macrophage from extracellular milieu; (6) Zn deprivation within yeasts may result from reduction in phagosomal

Zn; (7) MTs strongly sequester labile Zn in the macrophage, strictly denying metal access to H. capsulatum, thus depriving them of bioavailable Zn; (8) Labile Zn is routed away from the cytoplasm, into the Golgi apparatus in association with elevated Slc30a4 and Slc30a7; (9) The

Golgi apparatus is a „labile-Zn storage depot‟, which reserves the metal for Zn-dependent macrophage function; (10) GM-CSF increases proton channel Hvcn1 expression and MT-Zn sequestration which escalates HV1 function and phagosomal ROS generation via NADPH oxidase; (11) This strategic GM-CSF signaling cascade results in arrest of fungal growth. Solid lines, defined links; dotted lines, predicted links.

SUMMARY

Macrophages possess numerous mechanisms to combat microbial invasion, including sequestration of essential nutrients, like Zn. The pleiotropic cytokine granulocyte macrophage- colony stimulating factor (GM-CSF) enhances antimicrobial defenses against intracellular pathogens such as Histoplasma capsulatum, but its mode of action remains elusive. We have found that GM-CSF activated infected macrophages sequestered labile Zn by inducing binding to metallothioneins (MTs) in a STAT3 and STAT5 transcription factor-dependent manner. GM-

CSF upregulated expression of Zn exporters, Slc30a4 and Slc30a7; the metal was shuttled away from phagosomes and into the Golgi apparatus. This distinctive Zn sequestration strategy elevated phagosomal H+ channel function and triggered reactive oxygen species (ROS) generation by NADPH oxidase. Consequently, H. capsulatum was selectively deprived of Zn, thereby halting replication and fostering fungal clearance. GM-CSF mediated Zn sequestration via MTs in vitro and in vivo in mice and in human macrophages. These findings illuminate a

GM-CSF-induced Zn-sequestration network that drives phagocyte antimicrobial effector function.

INTRODUCTION

Zn is an abundant transition metal in living organisms. It is essential in immune homeostasis and function and Zn deficiency has been associated with thymic atrophy, impaired B, T and NK cell responses and T helper-1 (Th1) cytokine production9. Zn also functions in intracellular signaling68. Such a wide association with cellular functions requires strict regulation of Zn.

Availability of Zn to biomolecules is tightly regulated by binding proteins, metallothioneins (MTs) and Zn transporters. MTs are cysteine-rich proteins that bind up to seven

Zn ions with picomolar affinity and carry the labile Zn fraction to cellular compartments. MTs are induced during oxidative stress, scavenge reactive oxygen species (ROS) and reduce heavy metal intoxication26.

Macrophages provide a crucial role in immune defense; they phagocytose and kill intracellular pathogens by oxidative burst, nitric oxide production69 and T cell activation70.

„Nutritional immunity‟ signifies deprivation of essential nutrients to pathogens, thereby exerting an antimicrobial effect1. In this regard, interferon gamma (IFN-) deprives Fe in macrophages by regulating transferrin receptors and the transporter Slc11a158, 60. In contrast, macrophages may intoxicate Mycobacterium tuberculosis with Zn in phagosomes71. Zn competitively inhibits Mn binding to an essential virulence determinant in Streptococcus pneumoniae72, rendering it inactive. Thus, both metal starvation and intoxication are part of the arsenal employed by macrophages in host defenses.

Apart from directly exploiting metals for combating pathogens, immune cells also need them for activation of defense mechanisms. In this regard, modulation of Zn activates microglia12 and dendritic cells13 by altering surface markers and cytokine expression. Thus, Zn regulation is crucial in determining the outcome of an infectious process.

The fungal pathogen, H. capsulatum, is distributed worldwide and causes pulmonary and disseminated histoplasmosis, particularly in immunocompromised patients. For clearance of infection, coordinated action of innate and adaptive immunity is essential. The fungus gains entry into the host primarily via pulmonary route, is phagocytosed and replicates within resting macrophages73. Activation of macrophages with IFN-or GM-CSF promotes fungal growth inhibition. While IFN- acts only on murine macrophages in vitro, GM-CSF inhibits growth of yeasts in both murine and human macrophages47. A lack of GM-CSF is lethal during infection and treatment with recombinant GM-CSF accelerates fungal clearance53.

We have reported that Zn is essential for survival of H. capsulatum within macrophages and chelation causes drastic retardation of fungal growth45. Here, we elucidate a mechanism by which GM-CSF activates macrophages to preferentially sequester Zn while enhancing ROS production and denying the intracellular pathogen access to this element. Using a murine pulmonary model of H. capsulatum infection in vivo, we reveal the significance of GM-CSF in

Zn sequestration by MTs in infected macrophages. Finally, we have shown that this effect of

GM-CSF is conserved in human macrophages.

RESULTS

GM-CSF modulates total Zn and Zn proteome distribution

An unbiased inductively coupled plasma-mass spectrometry (ICP-MS) total elemental analysis was performed to evaluate changes in the concentration of Zn, Fe, Cu and Mn in macrophage lysates and H. capsulatum itself. The total Zn concentration increased in GM-CSF activated peritoneal macrophages compared to the resting control, and increased further in activated peritoneal and bone marrow macrophages during infection with 5 yeasts per macrophage. In contrast, Zn concentration was significantly reduced (p<0.001) in H. capsulatum recovered from activated macrophages in comparison to yeasts from resting peritoneal macrophages, and media- cultured H. capsulatum, suggesting that GM-CSF activation induced deprivation of yeast associated Zn. GM-CSF also altered Fe within macrophages, but not in intracellular yeasts.

Meanwhile, activation did not alter the concentration of Cu and Mn in macrophages and yeasts

(Figures 1A and 1B). Thus, GM-CSF specifically altered Zn flux in H. capsulatum infected peritoneal and bone marrow macrophages.

To examine the Zn-proteome distribution in macrophages, we used size exclusion chromatography based on molecular size via hydrodynamic radii coupled to ICP-MS (SEC-ICP-

MS). The Zn signal increased upon exposure to GM-CSF compared to resting peritoneal macrophages and a greater increase was seen upon activation and infection (Figure 1C). A large increment in the Zn signal was observed in infected activated bone marrow macrophages compared to uninfected control (Figure 1D). The relative Zn distribution between fractions was altered; the principal change occurred at ~20 min (≈20-7 kDa), representing a major portion of total Zn. This correlated with a reduction in the labile, exchangeable Zn pool appearing after 22 min in infected activated peritoneal and bone marrow macrophages (Figures 1E and 1F). Zn

26 distribution was altered dynamically as early as 2 h and held a similar trend for the 24 h time period in infected activated peritoneal macrophages (Figures S1A and S1B). To determine the specificity of GM-CSF in Zn modulation, macrophages were treated with the pro-inflammatory cytokine, tumor necrosis factor-α TNF-α). TNF-α did not alter the Zn signal in macrophages

(Figures 1G and 1H).

GM-CSF alters Zn transporter expression in H. capsulatum infected macrophages

We hypothesized that GM-CSF alters macrophage Zn distribution by regulating transporters. In an unbiased analysis of expression of Zn importers, ZIPs (Scl39a1-14) and exporters, ZNTs

(Slc30a1-10), Slc39a2 was upregulated in activated-infected peritoneal and bone marrow macrophages, by 60 and 40 fold respectively, compared to the control (Figures 2A and 2C). A significant 2-4 fold increase in Slc30a4 and Slc30a7 (p<0.001 for peritoneal and p<0.05 for bone marrow macrophages) was also observed upon activation and infection (Figures 2B and 2D).

The expression of another importer, Slc39a8 was unchanged (Figure S2A). TNF-α treatment of did not alter Slc39a2, Slc30a4 and Slc30a7 expression (Figure 2E). These data suggest that GM-

CSF activated-infected macrophages may increase Zn import and mobilize it into intracellular organelles via exporters.

Proteomics analysis of SEC fractions

GM-CSF altered the Zn-binding proteome in macrophages. For proteomics analysis, five fractions identified by SEC-ICP-MS were collected with the plasma turned-off and analyzed using a bottoms-up proteomics scheme. The list of identification parameters and proteins in all fractions and Zn-binding proteins highlighted in the fractions at ~10 and ~20 min are in Table

S1. The CCCH zinc finger protein, ZC3H12A, calreticulin and MT2 were selected as being potentially associated with immune response and Zn binding. MT2 was detected in the peak that scavenged a majority of Zn at 20 min. MT1, MT3 and ZIP2 were not identified in database search. ZIP2 is located on the cell membrane. The association of trans-membrane proteins with hydrophobic lipids and their insolubility poses a challenge in proteomic analysis74. Peptide matching is compromised by complex covalent hetero-oligomerization of MT isoforms, likely explaining the absence of MT1 and MT3 as hits in database search. We observed oligomerization at ~20 min based on a shift in retention time towards the high molecular weight region, with the peak showing maximal absorbance at 260 nm, typical of -S-S- bonds formed by

MTs during oligomerization, and not 280 nm, representative of Trp and Tyr containing proteins

(Figure S1C).

GM-CSF, but not TNF-induces MT expression in macrophages

GM-CSF enhanced Zn binding to the ~20 min fraction in infected macrophages (Figures 1C and 1D). Proteomic analysis and UV-Vis spectral characteristics suggested a major contribution of MTs to this Zn signal (Figure S1C, S1D, and S1E). Therefore, we examined the effect of

GM-CSF on Mt expression. GM-CSF, but not TNF-α increased Mt1, Mt2 and Mt3 expression in macrophages during infection (Figures 2F and 2G), Mt3 is predominantly expressed in the brain75.To ensure that our findings were valid, Mt3 was examined in hepatocytes and thymocytes and was not upregulated (Figure S2B). Expression of the Zn finger ribonuclease,

Zc3h12a was enhanced in infected macrophages but was not specific to GM-CSF (Figures S2D and S2E). GM-CSF did not alter the expression of calreticulin and of the metal response element

28 binding transcription factor-1 (Mtf1) which regulates Mt expression (Figure S2C and S2F).

Thus, GM-CSF specifically induced Mt expression.

GM-CSF mobilizes labile Zn away from H. capsulatum

Confocal microscopy was used to visualize labile Zn with the fluorescent probe, Zinpyr-176.

Uninfected resting peritoneal macrophages showed labile Zn distribution in the cytoplasm and epinuclear space (Figure 3A); however, Zn staining was diffuse in infected resting peritoneal macrophages (Figure 3C). GM-CSF activation caused focal Zn accumulationFigures 3B and

3Fand mobilized Zn away from H. capsulatumFigures 3D and 3G into the Golgi apparatus

(Figure 3I). The reduced Zn fluorescence of yeasts observed in GM-CSF activated macrophages was not due to an impermeability of the dye (Figure S3A). Previous reports suggest that Zinpyr-

1 may localize in the Golgi77. To ensure that labile Zn localization was a result of cytokine stimulation, we evaluated it in TNF-α treated macrophages. Zn distribution was non-focal, no or very little Zn staining was observed in the Golgi and labile Zn was observed in yeast (Figures

3E and 3H). This is consistent with lack of changes in Zn regulation in TNF-α treated macrophages (Figures 1G, 1H, 2E and 2G). Peritoneal macrophages treated with Zn and the ionophore, pyrithione, yielded bright fluorescence and very dim staining was observed in cells treated with the Zn chelator N,N,N‟,N‟-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN)

(Figures S3B and S3C). Labile Zn was examined in alveolar macrophages, the initial phagocyte that encounters H. capsulatum in vivo. While staining occurred in the cytosol and Golgi in resting alveolar macrophages, GM-CSF activation caused Zn localization in the Golgi (Figures

S3D-S3G). Collectively, these data suggest that GM-CSF induced Zn redistribution in macrophageswhich may limit its accessibility to H. capsulatum.

Metallothioneins sequester and deprive H. capsulatum of Zn

Since Zn regulation was similar in both types of macrophages, we utilized bone marrow macrophages for further studies. To determine if Zn sequestration in macrophages and deprivation in H. capsulatum was caused by MTs, we took two approaches. First, we silenced the expression of all four Mt genes (Figure 4A). Mt-silenced macrophages manifested reduced

Zn binding to the chromatographic fraction at 20 min accompanied by an increase in the labile

Zn fraction at ~25 min. (Figure 4B and zoom in). H. capsulatum recovered from Mt silenced macrophages had higher Zn content (p<0.01) (Figure 4C). Next, we examined Zn sequestration in Mt1-/-Mt2-/- macrophages. These cells exhibited a lack of Zn-binding to the fraction at 20 min, accompanied by an increase in labile Zn in GM-CSF treated and infected macrophages (Figure

4D and zoom in). H. capsulatum recovered from Mt1-/-Mt2-/- macrophages had higher Zn concentration (p<0.01) compared to wild type (WT) cells (Figure 4E). Thus, Zn deprivation within H. capsulatum resulted preponderantly from sequestration by MTs.

We queried if MT1 and MT2 participated in antimicrobial defense. We postulated that the lack of Zn sequestration in the absence of MT1 and MT2 would diminish the ability of GM-

CSF to inhibit fungal growth. While H. capsulatum growth was inhibited in WT activated macrophages, Mt1-/-Mt2-/- activated cells showed decreased ability to limit growth of yeasts

(Figure 4F).

GM-CSF engages the STAT5 and STAT3 transcriptional program to mediate Zn regulation

We sought to determine the signaling mechanism that resulted in Zn modulation. GM-CSF signals through STAT551 and STAT binding sites are present on the Mt promoter30. Inhibition of

STAT5 and STAT3 reduced MT-Zn binding (Figure 4G and 4H). To confirm our observations with chemical inhibitors, we silenced Stat5 and Stat3, which decreased Zn binding to MTs in the chromatograms of activated infected macrophages (Figures 4I, 4J and 4K).

These findings led us to postulate that GM-CSF may directly activate STAT5 and STAT3 signaling. GM-CSF increased STAT3 phosphorylation 10 min post-activation and infection, proving that the cytokine directly activated STAT3 (Figure 4L). There was >2.5 and >5 fold increase in Mt1 and Mt2 expression, as early as 10 min post infection and remained high for 24 h; Mt3 was modestly altered (Figure 5A). These data indicate that Zn regulation by GM-CSF occurs via STAT5 and STAT3 signaling.

We investigated the importance of Slc39a2 in regulating GM-CSF function. Activated infected macrophages manifested a ~10 fold increase in Slc39a2 expression 10 min post infection (Figure 5A). We questioned whether the regulation of Slc39a2 was a prerequisite in macrophage defense. Upregulation of Mt genes was partially dependent on Slc39a2, but silencing Slc39a2 did not alter Zn deprivation in H. capsulatum, ROS production or growth inhibition (Figures 5B, 5C, S4A and S4B). Slc39a2 silencing reduced Zn influx and MT-Zn signal in macrophages, but did not affect labile Zn sequestration (Figures 5C and 5D). To probe how MTs in these macrophages established a defensive state against the pathogen, we compared the amount of Zn bound per MT by determining the [Zn] to [S] ratio (Zn2+ to MTs) between control and Slc39a2 silenced macrophages. As expected, scramble siRNA treated macrophages had increased MT and sequestered labile Zn (Figure 5D). But, the amount of Zn bound to MTs in Slc39a2 silenced infected macrophages was ~2.4 fold greater than scramble siRNA treated control, thereby enabling efficient Zn sequestration (Figure 5E), even with lower production of

MTs. Silencing Slc39a14 did not affect GM-CSF driven macrophage defense (Figure S4C-F).

Zn sequestration enhances oxidative burst

GM-CSF enhances ROS production78. Therefore, we asked if Zn sequestration affected oxidative burst during infection. GM-CSF increased ROS in macrophages. Zn chelation by TPEN in serum-free medium augmented ROS production by ~40%, which was significantly higher

(p<0.05) compared to activated infected macrophages (Figure 6A). This effect was reversed upon addition of ZnSO4 (p<0.001). To ensure that this was not due to chelation of other metals by TPEN, we specifically depleted Zn from serum-free and serum-containing media. Low-Zn serum containing media did not show a significant increase in ROS over activated infected macrophages (Figure 6A), possibly due to the presence of residual Zn contributed by serum or serum-LPS that may have an inhibitory effect on NADPH oxidase (Nox) activity. However, macrophages exposed to Zn-depleted serum-free media manifested ~36% increase in ROS, significantly higher (p<0.01) than activated infected macrophages (Figure 6A). These data emphasize that a labile Zn-deprived state induced by GM-CSF facilitated increased oxidative burst, which was entirely reversible upon addition of Zn.

The sources of cellular ROS include mitochondrial respiration79 and ROS produced by

Nox80. Nox contributes to phagosomal ROS production that targets pathogens contained within these vacuoles. Activated macrophages from Ncf1-/- mice failed to generate an increased ROS response upon H. capsulatum challenge or Zn depletion (Figure 6B), indicating that reduction in labile Zn specifically enhanced ROS generation via Nox. We postulated that ROS production would be compromised in Mt1-/-Mt2-/- macrophages as a consequence of reduced Zn sequestration. While WT activated infected macrophages effectively induced an oxidative burst, this was dampened in Mt1-/-Mt2-/- macrophages (Figure 6E).

ROS was essential for H. capsulatum growth inhibition; macrophages in which Nox was inhibited by apocynin and Ncf1-/- macrophages had decreased ability to inhibit H. capsulatum growth (Figure 6C). To discern whether the defensive action of GM-CSF was a direct result of

Zn limitation or enhanced ROS caused by Zn deprivation, we subjected H. capsulatum to ROS in vitro under Zn limitation. Though xanthine oxidase (XO) at a higher concentration reduced growth in normal media, Zn limitation greatly enhanced the inhibitory effect of ROS on H. capsulatum growth and this was reversible upon addition of Zn (Figure 6D). To determine the mechanism by which Zn sequestration facilitated increased Nox activity, we examined the regulation of hydrogen voltage gated proton channel (Hvcn1). Macrophages enhanced Hvcn1 expression in response to GM-CSF in a STAT3 and STAT5 dependent manner (Figures 6F and

S5A). Proton flux by HV1 accelerates ROS generation via Nox and Zn inhibits HV1 function81.

Addition of the protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) reversed the inhibitory effect of Zn on ROS generation (Figure 6G). ROS production was attenuated in

Hvcn1 silenced and Hvcn1-/- macrophages upon GM-CSF activation and infection. Hvcn1-/- activated macrophages showed compromised ability to control H. capsulatum growth compared to WT control (Figures 6G, 6H, S5B, S5C). These data signify the impact of Zn sequestration in enhancing phagocyte defense via ROS production.

GM-CSF drives Zn binding to MT in vivo

GM-CSF has a profound influence on the fate of infection in vivo53. We hypothesized that GM-

CSF signaling was required for Zn sequestration in macrophages in vivo. In a pulmonary model of H. capsulatum infection, F4/80+ CD11b+ GFP+ and F4/80+ CD11b+ GFP- macrophages were sorted from lung leukocytes of green fluorescent protein (GFP) H. capsulatum infected mice

(Schematic, Figure 7A). F4/80+ CD11b+ GFP+ macrophages showed enhanced Zn binding to

MTs compared to F4/80+ CD11b+ GFP- cells(Figure 7B and S6G)To evaluate the importance of GM-CSF in Zn binding to MTs in vivo, it was neutralized and Zn profiles of macrophages were evaluated 7 days post infection (p.i). There was a sharp reduction in Zn bound to MTs in

F4/80+ CD11b+ GFP+ macrophagesin GM-CSF neutralized mice making it comparable to

F4/80+ CD11b+ GFP- macrophages(Figure 7D) No alteration was seen in Rat IgG controls

(Figure 7C). We asked whether GM-CSF neutralization enhanced fungal burden in macrophages. The percent and MFI of GFP+ cells in F4/80+ CD11b+ macrophages were higher in anti-GMCSF treated mice (Figure 7F). To further examine the importance of GM-CSF signaling in Zn regulation, WT macrophages were adoptively transferred into Csf2ra-/- mice lacking the

GM-CSF receptor, infected with GFP H. capsulatum, and WT and Csf2ra -/- macrophages were sorted 7 days post infection. Mt1, Mt2 and Slc39a2 expression was enhanced in infected WT macrophages, while Csf2ra-/- macrophages showed over 60, 93 and 97% decrease in Mt1, Mt2 and Slc39a2 expression respectively compared to infected WT macrophages (Figures 7G and

7H). Moreover, the percent infectivity of F4/80+ CD11b+ GFP+ cells in Csf2ra-/- mice was higher

(16.7%) as compared to infected WT CD45.1 macrophages (1.26%). These data highlight the importance of GM-CSF in regulating Zn during infection in vivo.

GM-CSF mediates Zn sequestration in macrophages from Slc11a1+/+ mice and from humans

C57BL/6 mice used in this study lack a functional SLC11A1 protein82. This prompted us to determine if Zn regulation was altered in the presence of this Fe transporter. GM-CSF induced a similar Zn profile in macrophages from CBA/J mice that express functional SLC11A1 (Figure

S6A-S6C). We asked if this phenomenon occurred in human macrophages. Zn distribution was analyzed in GM-CSF activated infected human macrophages compared with resting control.

There was a similarity between Zn profiles in human and murine macrophages. Activation of human macrophages with GM-CSF elevated MT-Zn binding during infectionFigures 7E and

S6H. These data indicate that GM-CSF broadly induces modulation of Zn in macrophages and that this is a conserved mechanism between mice and humans. We also determined if GM-CSF promoted Zn sequestration to a distinct clade of H. capsulatum, G186R. Activated macrophages strongly induced Zn modulation via MTs and Zn transporters upon infection with G186R H. capsulatum (Figure S6D-S6F). Thus, Zn sequestration by GM-CSF arrests pathogen survival by dually targeting fungal Zn acquisition and augmenting oxidative damage to the fungus.

DISCUSSION

This study revealed an underlying mechanism for the action of GM-CSF in combating infection with an intracellular pathogen. To date, the mechanism of GM-CSF function has not been fully elucidated. We have demonstrated that GM-CSF signaling empowers macrophage defense by facilitating a dual outcome of limitation of bioavailable Zn and simultaneous enhancement of superoxide burst which renders the pathogen susceptible to the host.

During infection, GM-CSF reduced total Zn in yeasts, but enhanced Zn influx in macrophagesAlthough this finding appears counterintuitive, GM-CSF induced an increase in the Zn binding proteome suggesting an enhancement of Zn dependent cellular functions such as transcription, enzyme function and metabolism83. To cope with pathogen invasion, activated macrophages sequestered the exchangeable Zn fraction away from intracellular fungus, while concurrently increasing ROS production.

We have reported that GM-CSF decreased total Zn in macrophages45. The discrepancy between the current and past study is a result of an improvement in the analytical method, specifically in the blank correction of lysate analysis. We went from a flow injection analysis in a metal containing system, to a continuous flow analysis in a metal free system. The SEC-ICP-

MS chromatograms manifest the same results in both studies with more total Zn in GM-CSF activated and infected peritoneal and bone marrow macrophages than the resting states by calculation of area under the curve. Also, total Zn in H. capsulatum was consistent, since the metal free system was used in both studies. Thus, the observation that GM-CSF activation induces a state of Zn sequestration and reduces total Zn in H. capsulatum has been reproducibly demonstrated in this study.

Confocal microscopy revealed that GM-CSF, but not TNF-α treated macrophages route labile Zn into the Golgi, in association with increased expression of Slc30a4 and Slc30a7 transporters that channel Zn into this organelle22. Zn deprivation in H. capsulatum was unique to

GM-CSF activated macrophages demonstrating the specificity of this cytokine in Zn mobilization. The Golgi acts as a site of labile Zn storage84, or a “Zn-storage depot” in restricting

Zn access to intracellular pathogens, while reserving it for phagocyte function. Fluorescent Zn probes are an invaluable tool for imaging Zn, but their application is affected by signal variation due to pH, ionic strength and propensity to localize in acidic compartments85. While total cellular

Zn is in micromolar range, the labile Zn pool identified using Zinpyr-1 is in the picomolar range17. Hence, the observed Zn signal may represent a fraction of total Zn. Nevertheless, labile

Zn is a crucial component of the total Zn pool. Immune cells deliver Zn signals upon pathogen contact and changes in labile Zn pool may reflect signal transducing properties of this metal ion68.

Our observations with Mt silenced and Mt1-/-Mt2-/- macrophages reinforce the critical function of MTs in antimicrobial defenses. In activated macrophages, MTs powered the loss of pathogen-associated Zn. We demonstrate that MTs are essential mediators of GM-CSF function and their absence presents a survival advantage to the pathogen. Of note, the WT control used in

Mt1-/-Mt2-/- studies (SvJ strain) depicted a Zn regulatory response similar to C57BL/6 macrophages. Thus, interference with MT regulation impairs the antimicrobial effect induced by

GM-CSF.

We showed that both STAT5 and STAT3 orchestrate Zn modulation guided by GM-CSF.

Disruption of STAT5 and STAT3 signaling abrogated MT-Zn sequestration. STAT5 and STAT3 heterodimerize in the presence of granulocyte-colony stimulating factor86; in this context, GM-

CSF may also induce heterodimerization to regulate MT expression. We sought to explain Zn alteration in infected macrophages by examining transporters. ZIP2 imports Zn into the cytosol from the extracellular milieu17. Slc39a2 silencing reduced MT expression, but did not affect the ability of MTs to sequester labile Zn. Upregulation of Slc39a2 may indicate its involvement in cellular processes that do not directly affect macrophage defense functions within the analyzed

24h period of infection. It is also plausible that the pathogen triggers Zn influx via ZIP2, an effect, counteracted by Zn sequestration in a GM-CSF primed macrophage.

Our findings reveal that Zn sequestration exerts a broader impact on macrophage antifungal defense. Conflicting evidence exists regarding the ability of Zn to trigger or dampen

ROS production81, 87. In activated macrophages, a reduction in the labile Zn pool was clearly associated with an increase in ROS that was dependent on Nox and reversed by exposure to Zn.

GM-CSF achieved this feat by a two-fold regulatory mechanism: first, the cytokine upregulated expression of Hvcn1 that elevates Nox activity; second, it induced a Zn limiting environment, that promotes proton pumping by HV1 and Nox function. Ncf1-/- and Hvcn1-/- activated macrophages exhibited reduced growth inhibitory effect on H. capsulatum. Apocynin inhibits

Nox, but may also interfere with NO production, and that may explain the increased reversal of inhibition88. Charge compensation by K+ channels may account for the partial growth inhibitory capacity seen in Hvcn1-/- activated macrophages89. An increased ROS triggers a variety of second messenger signals, such as activation of STAT390 and nuclear factor kappa B91 that possibly converge into potent and sustained macrophage defense. From the pathogen perspective, Zn paucity may compromise microbial protein functions such as superoxide dismutase activity that mediates resistance to the host92. Metal binding sites on proteins may exhibit degeneracy93. Zn limitation to the pathogen can therefore, potentially enforce substitution

38 of Zn dependent metal binding sites on microbial proteins with more reactive metals, such as Cu and Fe. Indeed, the ROS generated in vitro by XO exerted a profound effect on H. capsulatum survival under Zn limitation.

Emphasizing the impact of Zn sequestration on ROS generation, we demonstrated that

MTs are essential for an optimal ROS response induced by GM-CSF. In the absence of MT1 and

MT2, GM-CSF failed to effectively elevate ROS. These studies were performed at an early stage, because GM-CSF triggers MTs immediately following infection. This observation is surprising, since MTs bind metals but also scavenge ROS27. However, GM-CSF activated macrophages remarkably elevated Zn sequestration by MTs. The oligomeric shift in MTs may result from oxidation of Cys residues only during later stages of infection.

We have reported that lack of GM-CSF leads to detrimental fungal burden53. In this study, we found that blockade of GM-CSF in vivo was sufficient to diminish Zn sequestration and elevate macrophage fungal burden, signifying that among several cytokines, GM-CSF uniquely modulates MT-Zn homeostasis. This phenomenon may play a central role in inhibition of fungal growth in vivo. These mechanisms are conserved among different clades of H. capsulatum, among strains of mice and in humans.

In view of diversity in defense mechanisms against pathogens, human macrophages employ a unique Zn-intoxication strategy to limit growth of M. tuberculosis. Labile Zn deprivation by GM-CSF reveals a mechanism by which the host may combat intracellular pathogens. With these contrasting mechanisms of Zn intoxication versus deprivation, it can be postulated that cytokine-activated macrophages evolved to use specialized and distinct defense arsenals against different pathogens. On the other hand, microbes have evolved means to cope with nutrient paucity. Salmonella typhimurium resists calprotectin mediated Zn deprivation by

39 upregulating a high affinity Zn transporter94. In contrast, H. capsulatum fails to cope with Zn sequestration and enhanced free radical challenge. Little is known about Zn transporters in H. capsulatum, but one may derive that, while the organism strives to acquire Zn by increasing import, the amount is insufficient to drive intracellular growth. Of note, H. capsulatum does not have to confront an elevated calprotectin in macrophages45. On the other hand, it has to compete for Zn acquisition with MTs, which may be more potent than calprotectin in that each molecule of the former scavenges 7 ions with picomolar affinity26, while the latter binds 2 Zn ions per molecule with nanomolar affinity92, 95.

In summary, using a combination of gene expression analysis, microscopy, ICP-MS, SEC coupled to ICP-MS and proteomics, we have dissected a mechanism by which GM-CSF activated macrophages pose a dual defense system simultaneously challenging microbial tolerance to restricted Zn availability coupled to increased oxidative damage. Lastly, we propose that labile Zn deprivation may be a global defense strategy that can be extended to growth restriction of other intracellular fungi. Whether macrophages preferentially employ labile Zn deprivation over Zn intoxication for defense against distinct microbial classes remains to be determined.

EXPERIMENTAL PROCEDURES

See also supplementary procedures.

Mice

C57BL/6, CBA/J, Mt1tm1BriMt2tm1Bri (Mt1-/-Mt2-/-), 129S1/SvImJ WT (control for Mt1-/-Mt2-/-) and

B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) mice were obtained from Jackson Laboratory. We thank

Dr. Bruce Trapnell, Cincinnati Children‟s Hospital Medical Center for the Csf2ra-/- mice; Dr.

John Engelhardt, Dr. Yulong Zhang (University of Iowa), Dr. Brahm Segal, Dr. Nazmul Khan

(Roswell Park Cancer Institute) and Dr. Long-Jun Wu and Dr. Jiyun Peng (Rutgers University) for providing bone marrow from Ncf1-/- and Hvcn1-/- mice. Animals were maintained by the

Department of Laboratory Animal Medicine, University of Cincinnati, accredited by the

American Association for Accreditation of Laboratory Animal Care (Frederick, MD) and animal experiments were in accordance with Animal Welfare Act guidelines of National Institutes of

Health.

Activation, STAT inhibition and infection

Bone marrow macrophages were activated with cytokines for 24 h before infection. Peritoneal macrophages were rested for 24 h, followed by activation for 24 h before infection and were activated again during infection. Where indicated, macrophages were cultured throughout with

100µM STAT3 or STAT5 inhibitors, S3I-201 and N′-((4-Oxo-4H-chromen-3-yl)methylene) nicotinohydrazide (Calbiochem) respectively. For infection, G217B or G186R H. capsulatum were cultured in Ham‟s F12 (Zn concentration, 250 ± 18 ng/ml or 4 µM) and washed before use.

Microscopy

Macrophages were infected with PKH-26 stained H. capsulatum for 24 h followed by staining with nuclear, Golgi and Zn dyes where indicated for 30 min. Images were acquired on a Zeiss

LSM710 confocal and analyzed using ZEN 2011 software.

Silencing

Genes were silenced using TransIT-TKO transfection reagent (Mirus Bio LLC) and 100nM Mt

(Santa Cruz), Slc39a2, Slc39a14, Stat3 or Stat5 (50nM each of Stat5a and Stat5b), Hvcn1 or scrambled siRNA (Dharmacon) as per manufacturer‟s instructions.

ROS

Macrophages were activated for 24h and then incubated with 5µM Dihydroethidium (DHE)

(Invitrogen) in fresh media for 15 min in dark, followed by GM-CSF and infected for 2h. Where indicated, 100µM ZnSO4 and TPEN or Zn depleted media was added 20 min prior to DHE.

CCCP was added 30 min before analysis by flow cytometry (Accuri C6). Data were analyzed with FCS Express 3 De Novo Software (CA) or FlowJo.

In vivo

Eight week old C57BL/6 mice were infected with 2 X 106 GFP+ H. capsulatum yeasts i.n. Mice were injected ip. with 0.5mg control mAb Rat IgG2a (BioXcell) or MP1–22E9 mAb Rat IgG2a anti-GM-CSF a day before and on the day of infection. After 7 days, lungs were homogenized with gentleMACS Dissociator (Miltenyi Biotec, CA), collagenase treated, filtered through 60

µM nylon mesh and washed. Leukocytes were isolated using Lympholyte M (Cedarlane

Laboratories, Canada) and stained with APC F4/80 (AbD Serotec) and PerCP CD11b mAbs (BD

Biosciences) and CD16/CD32 blocking mAbs for 30 min at 4°C and washed; gated on

F4/80+CD11b+ and sorted into GFP+ (containing phagocytosed H. capsulatum) and GFP- (not containing H. capsulatum) using 5-laser FACS Aria II (BD Biosciences) in a BSLII facility at

Cincinnati Children‟s Hospital; collected at 4°C in culture media and washed prior to lysis. For adoptive transfer, 2X106 bone marrow macrophages from WT (CD45.1) were transferred

42 intratracheally into 8 week old Csfr2a-/- (CD45.2) mice 1 day prior to H. capsulatum infection.

On day 7 post infection, lung leukocytes were sorted into 4 populations:

F4/80+CD11b+CD45.1+GFP- or GFP+ and F4/80+CD11b+CD45.1-GFP- or GFP+ macrophages for analysis of gene expression.

Human macrophages 

PBMCs were isolated from human blood (Hoxworth Blood Center, University of Cincinnati) using Ficoll-Paque. Monocytes were purified with Monocyte Isolation Kit II on a VarioMACS separator (Miltenyi Biotec) and cultured in RPMI with 12.5% human male AB serum (Sigma-

Aldrich) for 7 days, and macrophages were exposed to 10 ng/ml GM-CSF (Peprotech) or vehicle for 24 h before infection, re-exposed to GM-CSF at the time of infection and lysates were prepared 24 h later. Human experiments were approved by the Institutional Review Board of the

University of Cincinnati.

Cell lysates for ICP-MS

Macrophages were washed and lysed with 0.1% sodium dodecyl sulfate (SDS) in HPLC water for 20 min on ice. Lysates were passed through a 0.22 µm filter or centrifuged at 13,000rpm for

5 min for analysis. For total metal analysis of H. capsulatum, lysates were centrifuged at

13,000rpm for 5 min to pellet the yeasts and the pellet was washed twice with HBSS.

Total elemental analysis and SEC-ICP-MS screening

ICP-MS analysis was performed on Agilent 7700x ICP-MS instrument (Agilent Technologies).

A conventional Meinhard nebulizer, Peltier-cooled spray chamber, and shield torch constituted the sample introduction system under standard conditions. Sc was used as internal standard and

SRM (DORM-3 or NIST1745) were analyzed for every digestion.

For SEC-ICP-MS, Agilent 1100 series HPLC system equipped with a binary pump, vacuum membrane degasser, thermostated auto sampler, column oven, and diode array detector (DAD), with a semi-micro flow UV-Vis cell was coupled to the ICP-MS through a 0.17mm internal diameter short PEEK tube. The system was controlled with Chemstation software. A TSK Gel

3000SW 7.5 x 300 mm column was used. For proteomics, samples were concentrated before injection using MWCO filter 3 kDa (Millipore). For analysis of MT saturation, the MTs-SEC signal (20 min) was collected and analyzed for S and Zn to determine the MT:Zn ratio in a

Superdex peptide SEC-ICP-MS using O2 reaction mode.

Proteomics

The SEC-ICP-MS fractions were analyzed using an Agilent 1200 nanoHPLC, nanoChip ESI and

6300 series MSD Ion Trap XCT Ultra system. Peptide and corresponding protein identification were conducted using MASCOT server (Matrix Science).

Statistics p-values were calculated using one way ANOVA for multiple comparisons and adjusted with

Bonferroni‟s or Holm Sidak correction; and non-paired Student t test where two groups were compared; *p<0.05; **p<0.01; ##p<0.001; NS, not significant.

ACKNOWLEDGEMENTS

We thank Agilent Technologies for instrumentation, ICP-MS and nanoHPLC-ESI-IT-MS and

Drs. B. Klein, W. Nauseef, and D. Hildeman for their valuable feedback. This work was supported by grants from the NIH, AI-094971, AI-106269 and a Merit Review from Veterans

Affairs.

AUTHOR CONTRIBUTIONS

K.S.V and J.L.F. contributed equally, designed and performed experiments, analyzed data and wrote the manuscript, K.S.V performed in vitro, microscopy, in vivo and human experiments and interpreted data, J.L.F. performed chromatographic separations with UV-Vis, ICP-MS and ESI-

IT-MS-MS analysis and interpreted data, A.P. performed bioinformatics analysis, J.C. and

G.S.D. designed and supervised the work. All authors participated in final manuscript preparation.

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

FIGURE LEGENDS

Figure 1: GM-CSF distinctly modulates Zn-distribution in infected macrophages

Total concentration of Zn by ICP-MS in (A, B) lysates from peritoneal and bone marrow macrophages RP, resting peritoneal; Act, GM-CSF activated, and, total Zn analysis by ICP-MS in H. capsulatum recovered from macrophages and compared with yeasts grown in Ham‟s F12 media, data are mean ± SD, 4 independent experiments. The cell lysate concentrations are based on 2.5 X 106 cells in ~200 µl; H. capsulatum concentration was calculated based on mass of yeasts recovered; (C, D) typical SEC-ICP-MS chromatograms of activated peritoneal and bone marrow macrophages, compared to resting conditions; (E, F) Left panel, Graphical representation of color coded peaks depicting Zn distribution in various MW fractions in the chromatograms of infected activated peritoneal and bone marrow macrophages; middle panel, Zn distribution calculated as area under the curve of individual peaks against the total area; red and green boxes with **, ## symbol indicate significant differences in the corresponding color coded fractions between different groups; data are mean ± SD, 4 independent experiments; (G, H)

Typical SEC-ICP-MS chromatograms of TNF-α treated macrophages, 3 independent experiments, Y axis in all chromatograms is off-set to allow easy comparison under the same scale. Related to Figure S1 and Table S1.

Figure 2: GM-CSF alters Zn transporter and MT expression  qRT-PCR of Slc39a and Slc30a in (A, B) peritonealC,D) bone marrow(E) TNF-α treated macrophages data are mean ± SEM, 3 independent experiments; (F, G) Mt1, Mt2 and Mt3 expression in macrophages RP, resting peritoneal; Act, GM-CSF activated; Hc, H. capsulatum,, data are mean ± SEM, 8 independent experiments; ND, not detected. Related to Figure S2.

Figure 3: GM-CSF, but not TNF-triggers Zn mobilization

Confocal microscopic analysis of Zn (green), nucleus (blue) and H. capsulatum (Hc) (red) in (A-

E) peritoneal; (F-H) bone marrow macrophages; merge, overlay of Zn, nucleus and H. capsulatum; (I) Staining for Zn (green), nucleus (blue) and Golgi (red); merge, overlay of Zn, nucleus and Golgi; yellow, co-localization of Golgi and Zn dyes; white arrows point at the Golgi; scale bars, 20µm; RP, resting peritoneal; P, peritoneal; Act, activated; 3 independent experiments. Related to Figure S3.

Figure 4: GM-CSF engages the STAT5 and STAT3 transcriptional program to regulate

MT-Zn sequestration

(A) qRT-PCR of Mt genes in activated (Act) macrophages treated with scramble or Mt siRNA,

% decrease in expression compared to scramble siRNA control, data are mean ± SEM, 2 independent experiments; (B, D) SEC-ICP-MS chromatograms of Mt silenced activated infected

(Hc) macrophages and from WT and Mt1-/ Mt2-/- mice and zoom in of labile Zn fraction, 2 independent experiments; (C, E) Total metal analysis of Zn in H. capsulatum (Hc) from Mt silenced and WT, Mt1-/-Mt2-/- macrophages, data are mean ± SD, 2 independent experiments; (F)

Colony forming units (CFU) of H. capsulatum 24 h post infection from WT and Mt1-/-Mt2-/- activated macrophages compared to untreated control; data are mean ± SEM, 4 independent experiments; (G-J) SEC-ICP-MS chromatograms of chemically inhibited and Stat3 and Stat5 silenced macrophages, % decrease in peak area is compared to scramble siRNA treated infected macrophages; Y axis, Offset Zn-signal, 3 independent experiments; (K) p-STAT3, p-STAT5 and

β-actin from silenced macrophages 24 h post activation, n=2; (L) Western blot of p-STAT3,

STAT3 and β-actin from lysates 10 min post activation and infection, n=2.

Figure 5: Early regulation of Mt genes and role of Slc39a2 in MT-Zn sequestration

(A) qRT-PCR of Mt1, Mt2, Mt3 and Slc39a2, time points are min and h post infection, data are mean ± SEM, n=3 at 0-12 h and n=10 independent experiments at 24 h; (B) qRT-PCR of

Slc39a2, Mt1 and Mt2 in Slc39a2 silenced macrophages normalized to scramble siRNA control,

% decrease in expression compared to respective scramble controls, data are mean ± SEM, 3 independent experiments; (C) Total metal analysis of Zn in lysates (left) and H. capsulatum

(right) from scramble siRNA and Slc39a2 silenced macrophages, data are mean ± SD, 3 independent experiments; (D) SEC-ICP-MS chromatogram of scramble siRNA and Slc39a2 silenced activated macrophages, Y axis, Offset Zn- signal; (E) Ratio of Zn to sulfur in the MT fraction collected from 19 to 21 min based on the areas under the chromatogram corrected by differences in sensitivity through calibration with S and Zn standards, data are mean ± SD, 3 independent experiments. Related to Figure S4.

Figure 6: Zn sequestration enhances ROS production

(A) % Change in ROS MFI in cells cultured in Zn replete, low Zn serum-, low Zn serum+, 10 M

- + + TPEN serum , 20 M TPEN serum , and 20 M TPEN with 100 M ZnSO4 treated serum media, serum- media was included as serum containing media has considerably higher Zn.

Where serum+ media was used, a higher concentration of TPEN was added (20M), last group is significantly different (p<0.01) from all groups; ROS histograms demonstrating the effect of Zn sequestration; Act, activated, Hc, H. capsulatum, 2-3 independent experiments; (B) % change in

ROS MFI in Ncf1-/- activated macrophages, compared to WT activated uninfected cells, 3 independent experiments, histogram from Ncf1-/- macrophages; (C) % inhibition of yeast growth in apocynin treated or Ncf1-/- activated over WT macrophages, 3 independent experiments; (D)

CFU of H. capsulatum in normal or low Zn media with increasing concentration of XO, with or without addition of ZnSO4, 3 independent experiments; (E) % change in ROS MFI in WT and

Mt1-/-Mt2-/- macrophages compared to WT uninfected cells, ROS histogram of WT and Mt1-/-

Mt2-/- macrophages, 3 independent experiments; (F) Hvcn1 expression normalized to untreated macrophages, 3 independent experiments; (G) % change in ROS MFI in WT activated

- macrophages in response to infection, ZnSO4 and CCCP, 2 independent experiments, and Hvcn1

/- activated cells compared to WT, 3 independent experiments; (H) % inhibition of H. capsulatum growth in activated WT vs Hvcn1-/- macrophages over WT control, 3 independent experiments; all data are mean ± SEM. Related to Figure S5.

Figure 7: GM-CSF triggers Zn binding to MTs in vivo and in human macrophages

(A) Schematic, mice were left untreated or treated with rat IgG or anti-GM-CSF and infected i.n. with 2 X 106 GFP+ yeasts (Hc); lungs were harvested 7 days p.i. and F4/80+CD11b+GFP+ and

F4/80+CD11b+GFP- cells were sorted. SEC-ICP-MS profiles of (B) F4/80+CD11b+GFP+ and

F4/80+ CD11b+GFP- cells isolated from lungs; (C) rat IgG treated and (D) anti-GM-CSF treated mice, 2 independent experiments; (E) SEC-ICP-MS profiles of lysates from human resting (h

R)and GM-CSF activated (h Act) macrophages; Y axis, offset Zn signal; inset, zoom in of 15-25 min fraction, 2 independent experiments; (F) Dot plot of lung leukocytes gated on

F4/80+CD11b+ macrophages; histogram of F4/80+CD11b+GFP+ macrophages; bar graphs are quantification of %, absolute numbers and MFI of F4/80+CD11b+GFP+ macrophages in rat IgG vs anti-GM-CSF treated mice, data are mean ± SEM, n=4; (G) Schematic representing transfer of CD45.1 WT macrophages into CD45.2 Csf2ra-/- mice, infection and sorting of WT and

Csf2ra-/- macrophages from lungs for gene expression analysis; (H) qRT-PCR of Mt1, Mt2 and

Slc39a2 from sorted CD45.1+ (WT) and CD45.1- (Csf2ra-/-) GFP- and GFP+ macrophages normalized to CD45.1+GFP- macrophages, % values show a decrement in gene expression in

Csf2ra-/- CD45.1- GFP+ macrophages compared to WT CD45.1+ GFP+ macrophages, data are mean ± SEM, data are from 1 experiment. Related to Figure S6.

Figure 1:

Figure 2:

Figure 3:

Figure 4:

Figure 5:

Figure 6:

Figure 7:

Immunity, Volume 39

Supplemental Information

Granulocyte Macrophage-Colony Stimulating Factor Induced Zn Sequestration Enhances

Macrophage Superoxide and Limits Intracellular Pathogen Survival

Kavitha Subramanian Vignesh, Julio A. Landero Figueroa, Aleksey Porollo,

Joseph A. Caruso, and George S. Deepe, Jr

Supplemental Inventory

1. Supplemental Figures and Tables

Figure S1, Related to Figure 1

Figure S2, Related to Figure 2

Figure S3, Related to Figure 3

Figure S4, Related to Figure 5

Figure S5, Related to Figure 6

Figure S6, Related to Figure 7

Table S1, Related to Figure 1

2. Supplemental Experimental Procedures

Figure S1:

Figure S1: Chromatographic and spectral behavior of MT-Zn signal in macrophages.

Related to Figure 1.

(A and B) Resting peritoneal (RP) macrophages and activated peritoneal (Act P) macrophages were infected with H. capsulatum (Hc), and SEC-ICP-MS analysis was performed on cell lysates collected at 2 - 24 h time intervals after infection, data are from one experiment; (C) SEC-ICP-

MS of activated infected bone marrow macrophages (black solid line), the fraction from 19-23 min was collected, denatured and analyzed showing a decrease in Zn signal (black dotted line); the collected fraction was treated with Cd, and analyzed again, showing a sharp Cd signal (blue line). The left axis corresponds to the Zn-related signal, and the right axis to the Cd-related signal. The 19-23 min peak corresponding to MTs undergoes a shift in retention time to the low molecular weight region upon treatment with Cd. The Cd signal represents de-oligomerized state

63 of MTs bound to Cd. The molecular weight in this region corresponds to the MTs in their monomeric state (6-7kDa), 2 independent experiments; (D and E) Contour map of UV-Vis absorbance (isoplots) of activated infected peritoneal and bone marrow (BM) macrophages from

210 to 500 nm (Y axis) and 0 to 30 min (X axis), at a response time of 5 spectra per second with a step of 2 nm, during the SEC-DAD-ICP-MS analysis of cell lysates. X axis, time in min; Y axis, wave length (λ), and each progressive line represents an increment of 5 mAu. The maximum of absorbance in each fraction can be seen as a round shape contour pattern, giving information of the composition of species eluted in the fractions. The chromatograms show maxima at two wave lengths, 280 nm, characteristic of proteins containing aromatic amino acids; and 260 nm, characteristic of nucleic acids (at HMM, first part of the chromatogram) and –S-S-,

-S-metal- complexes. The ~20 min region clearly indicates that the absorbance maximum is at

260 nm in all cases, while in the high molecular weight region of the chromatogram both, 280 and 260 nm maxima are observed; representative of 3 independent experiments.

Figure S2:

Figure S2: GM-CSF specifically alters Zn transporter and MT regulation. Related to

Figure 2.

(A) GM-CSF specifically targets the expression of Slc39a2, Slc30a4 and Slc30a7, because H. capsulatum (Hc) infected activated bone marrow macrophages (Act BM)do not alter expression of another importer, Slc39a8 over time. qRT-PCR of activated infected macrophages normalized to activated macrophagesSamples were collected at timed intervals post infection, data are mean ± SEM; NS, not significant, from 2 independent experiments; (B) qRT-PCR analysis of

Mt3 expression in thymocytes and hepatocytes of 6-8 week old male C57BL/6 mice, left untreated or treated with 50 ng/ml PMA + 500 ng/ml ionomycin or 10 ng/ml LPS E.coli 055: B5 were compared to resting peritoneal (RP) and activated peritoneal (Act PM) macrophages.

Values are normalized to resting peritoneal macrophage control, ND, not detected, data are mean

± SEM, n= 3 independent experiments; qRT-PCR on peritoneal and bone marrow macrophages reveals that GM-CSF either modestly or does not significantly alter the expression of (C) Mtf1;

(D, E) CCCH-zinc finger family members, Zc3h12a-d, (note that the change in Zc3h12a occurs irrespective of GM-CSF activation in peritoneal macrophages)and (F) calreticulin, data are mean ± SEM, NS, not significant, n=3 independent experiments.

Figure S3:

Figure S3: Sensitivity of Zinpyr-1 and GM-CSF driven Zn localization in alveolar macrophages. Related to Figure 3.

(A) H. capsulatum (Hc) cultured in Ham‟s F12 was stained as described in supplementary experimental procedures and imaged; merge, overlay of Zn, nucleus, and H. capsulatum, scale bar, 20 µm; Bone marrow macrophages were treated with (B) 2 µM TPEN overnight or (C) 100

µM ZnSO4 and 20 µM pyrithione for 30 min, stained with Zinpyr-1, data represent images obtained from 3 different fields; scale bar, µm; Labile-Zn staining of D, E resting alveolar

Rmacrophages and F, Gactivated alveolar (Act A)macrophagesbright field (left panel),

Zn (middle panel), merge (right panel); zoom images, scale bar, 20 µm; representative of 2 independent experiments.

Figure S4:

Figure S4: Role of Slc39a2 and Slc39a14 in GM-CSF function. Related to Figure 5.

(A) Growth inhibition of H. capsulatum (Hc) in scramble siRNA or Slc39a2 siRNA treated activated bone marrow (Act BM) macrophages compared to scramble-treated control; (B) % change in ROS MFI in scramble and Slc39a2 siRNA treated activated infected macrophages compared to scramble siRNA treated activated macrophages; (C) qRT-PCR analysis of Slc39a14 in activated infected macrophages treated with scramble or Slc39a14 siRNA, values are normalized to scramble treated activated macrophages, percent decrease in expression compared to scramble siRNA controls, 2 independent experiments; (D) SEC-ICP-MS analysis of Slc39a14 silenced activated and infected macrophages compared to scramble siRNA control, Y axis,

Offset Zn signal, 3 independent experiments; (E) Total Zn analysis by ICP-MS in cell lysates

69 from scramble and Slc39a14 silenced activated and infected macrophages; (F) Total Zn analysis by ICP-MS in yeasts from scramble and Slc39a14 silenced activated macrophages, data are mean

± SEM; 3 independent experiments.

Figure S5:

Figure S5: Regulation of Hvcn1 expression and its role in superoxide burst. Related to

Figure 6.

(A) qRT-PCR of Hvcn1 in Stat3 and Stat5 silenced activated and infected macrophages compared with scramble siRNA treated control, data are mean ± SEM, 2 independent experiments; (B) qRT-PCR of Hvcn1 in activated bone marrow (Act BM) macrophages treated with scramble or Hvcn1 siRNA and infected with H. capsulatum (Hc), % decrease in expression compared to scramble siRNA treated controls, 2 independent experiments; (C) % change in ROS

MFI in Hvcn1 silenced activated and infected macrophages compared to scramble siRNA treated activated macrophages, data are mean ± SEM, 3 independent experiments.

Figure S6:

Figure S6: Zn regulation by GM-CSF occurs across different strains of mice and clade of

H. capsulatum. Related to Figure 7. (A, B) qRT-PCR analysis of Mt1, Mt2, Mt3, Slc39a2,

Slc30a4 and Slc30a7 in Slc11a1+/+ activated bone marrow (Act BM) macrophages derived from

CBA/J mice and infected with H. capsulatum (Hc), data are mean ± SEM, 4 experiments; (C)

SEC-ICP-MS analysis of activated and infected macrophages from CBA/J mice, Y axis, Offset

Zn signal, data are mean ± SEM, 4 independent experiments; (D, E) qRT-PCR analysis of Mt1,

Mt2, Mt3, Slc39a2, Slc30a4 and Slc30a7 in activated macrophages infected with G186R H. capsulatum, data are mean ± SEM, 2 independent experiments; (F)SEC-ICP-MS analysis of activated macrophages infected with G186R H. capsulatum, Y axis, Offset Zn signal, 4 independent experiments; (G and H) UV-Vis isoplot from the SEC-DAD-ICP-MS analysis of cell lysates from GFP+ macrophages obtained by sorting lung leukocytes from in vivo infection studies and from human activated (h Act) infected macrophages. The ~20 min region as pointed by the arrow indicates that the absorbance maximum is at 260 nm in both cases, while in the high molecular weight region of the chromatogram both, 280 and 260 nm maxima are observed, 2 independent experiments.

Proteomics Report

Table S1: Summary of protein IDs obtained from proteomics experiments. Related to Figure 1.

The SEC fractions are as follows: F1, 9.5-10.5 min (≥ 600 – 400 kDa); F2, 10.6-14 min (400 –

100 kDa); F3, 14.1-16 min (100-80 kDa); F4, 16.1-19 min (80 – 20 kDa); F5, 19.1-23 min (20 –

1 kDa). min (20 – 1 kDa). The reported protein scores are a mathematical addition of the ion scores from independent peptides assigned to the same protein, in the same search. Ion score is a probabilistic measurement of the random coincidence of the submitted MS and MS/MS spectra with the theoretical correspondent spectra. It is defined by MASCOT as -10Act-LOG10(P), where P is the absolute probability. A probability of 10-20 of having a false positive thus becomes a score of 200.

Resting peritoneal macrophage + H. capsulatum Protein Score Fraction

Nonmuscle heavy chain myosin II-A [Mus musculus] 49 F1 Talin-1 [Mus musculus] 68 F1 A disintegrin and metalloproteinase with thrombospondin 57 F1 motifs [Mus musculus]

12-lipoxygenase [Mus musculus] 54 F2 Integrin beta-2 [Mus musculus] 38 F2 Disintegrin and metalloproteinase domain-containing protein 51 F2 17 [Mus musculus]

Serpin precursor [Mus musculus] 49 F3 ATP synthase subunit alpha, mitochondrial precursor [Mus musculus] 48 F3 Aldehyde dehydrogenase, mitochondrial [Mus musculus] 37 F3 Tubulin beta-5 chain [Mus musculus] 38 F3 Alpha-enolase [Mus musculus] 32 F3 Polyubiquitin-C [Mus musculus] 30 F3

Substance-P receptor [Mus musculus] 47 F3 Cathepsin D [Mus musculus] 41 F3 pyruvate kinase M [Mus musculus] 111 F3 cathepsin D precursor [Mus musculus] 67 F3 A-X actin [Mus musculus] 64 F3 gamma actin-like protein [Mus musculus] 64 F3 arginase-1 [Mus musculus] 52 F4 Pre-mRNA-splicing factor 18 [Mus musculus] 43 F4 Beta-enolase [Mus musculus] 242 F4 Rho GDP-dissociation inhibitor 1 [Mus musculus] 165 F4 Ras-related protein Rab-7a [Mus musculus] 82 F4 histone H4 (55AA) (1 is 3rd base in codon) [Mus musculus] 57 F5 Histone H4 [Mus musculus] 81 F5 ATP synthase subunit O, mitochondrial [Mus musculus] 34 F5 Cofilin-1 [Mus musculus] 30 F5

Activated peritoneal macrophage Filamin-A [Mus musculus] 290 F1 Plasminogen activator inhibitor 2, macrophage [Mus musculus] 43 F1 Tubulin alpha-1A chain [Mus musculus] 40 F1 Myosin-9 [Mus musculus] 93 F1 Talin-1 [Mus musculus] 140 F1

Vimentin [Mus musculus] 381 F2 Heat shock protein HSP 90-beta [Mus musculus] 375 F2 Heat shock protein 75 kDa, mitochondrial [Mus musculus] 375 F2 Endoplasmin [Mus musculus] 195 F2

Macrophage-capping protein [Mus musculus] 37 F3

60S ribosomal protein L4 [Mus musculus] 37 F3 Ubiquitin-like modifier-activating enzyme ATG7 [Mus musculus] 36 F3 Plastin-3 [Mus musculus] 78 F3 Arginase-1 [Mus musculus] 154 F3 Cathepsin Z [Mus musculus] 127 F3 Cathepsin S [Mus musculus] 112 F3 Alpha-enolase [Mus musculus] 105 F3 Adenylyl cyclase-associated protein 1 [Mus musculus] 95 F3 Cytosolic non-specific dipeptidase [Mus musculus] 59 F3 Actin-related protein 2 [Mus musculus] 44 F3 Pre-mRNA-splicing factor 18 [Mus musculus] 43 F3 Plastin-2 [Mus musculus] 96 F3

Transketolase [Mus musculus] 138 F4 Tubulin beta-5 chain [Mus musculus] 129 F4 Lysozyme C-2 [Mus musculus] 92 F4 Heterogeneous nuclear ribonucleoprotein K [Mus musculus] 89 F4 Ras-related protein Rab-7a [Mus musculus] 82 F4 Plastin-3 [Mus musculus] 78 F4 Septin-2 [Mus musculus] 71 F4 Beta-actin-like protein 2 [Mus musculus] 68 F4

Profilin-1 [Mus musculus] 71 F5 Ubiquitin-40S ribosomal protein S27a [Mus musculus] 243 F5 Fatty acid-binding protein, epidermal [Mus musculus] 224 F5

Activated peritoneal macrophage + H. capsulatum Vimentin [Mus musculus] 135 F1 Myosin-9 [Mus musculus] 126 F1

Pyruvate kinase isozymes M1/M2 [Mus musculus] 105 F1 Alpha-enolase [Mus musculus] 84 F1 Actin, cytoplasmic 2 [Mus musculus] 59 F1 Cathepsin D [Mus musculus] 51 F1 Histone H4 [Mus musculus] 44 F1 Actin, aortic smooth muscle [Mus musculus] 37 F1 Myosin-9 [Mus musculus] 148 F1 Myosin-10 [Mus musculus] 59 F1

60 kDa heat shock protein, mitochondrial [Mus musculus] 83 F2 14-3-3 protein zeta/delta [Mus musculus] 69 F2 Sulfated glycoprotein 1 [Mus musculus] 50 F2 Ribonuclease ZC3H12A [Mus musculus] 66 F2 Cofilin-1 [Mus musculus] 47 F2 Otoraplin [Mus musculus] 46 F2 Alpha-enolase [Mus musculus] 45 F2 Plastin-2 [Mus musculus] 68 F2 Integrin beta-2 [Mus musculus] 41 F2 78 kDa glucose-regulated protein [Mus musculus] 95 F2

Galectin-3-binding protein [Mus musculus] 54 F3 Protein disulfide-isomerase A6 [Mus musculus] 51 F3 ATP synthase subunit alpha, mitochondrial [Mus musculus] 47 F3 Integrin beta-2 [Mus musculus] 41 F3 T-complex protein 1 subunit theta [Mus musculus] 40 F3 Vimentin [Mus musculus] 142 F3 Alpha-enolase [Mus musculus] 114 F3 Pyruvate kinase isozymes M1/M2 [Mus musculus] 90 F3 Protein disulfide-isomerase [Mus musculus] 85 F3

Creatine kinase B-type [Mus musculus] 71 F3 Transmembrane glycoprotein NMB [Mus musculus] 67 F3 Beta-enolase [Mus musculus] 66 F3 Alpha-2-macroglobulin receptor-associated protein [Mus musculus] 58 F3 Malate dehydrogenase, mitochondrial [Mus musculus] 58 F3 Fructose-bisphosphate aldolase A [Mus musculus] 56 F3 Alcohol dehydrogenase [NADP+] [Mus musculus] 56 F3 Aldehyde dehydrogenase, mitochondrial [Mus musculus] 53 F3 Tropomyosin alpha-4 chain [Mus musculus] 51 F3 60 kDa heat shock protein, mitochondrial [Mus musculus] 49 F3 Coiled-coil domain-containing protein 38 [Mus musculus] 42 F3

Calreticulin [Mus musculus] 42 F3 Sulfated glycoprotein 1 [Mus musculus] 187 F3 Alpha-enolase [Mus musculus] 175 F3 Cathepsin S [Mus musculus] 154 F3 Actin, cytoplasmic 1 [Mus musculus] 110 F3 Annexin A1 [Mus musculus] 110 F3 Beta-actin-like protein 2 [Mus musculus] 89 F3 ATP synthase subunit alpha, mitochondrial [Mus musculus] 73 F3 78 kDa glucose-regulated protein [Mus musculus] 66 F3 Cathepsin B [Mus musculus] 60 F3 Peroxiredoxin-4 [Mus musculus] 57 F3 Malate dehydrogenase, mitochondrial [Mus musculus] 46 F3

Ganglioside GM2 activator [Mus musculus] 52 F4 Malate dehydrogenase, mitochondrial [Mus musculus] 45 F4 Peroxiredoxin-1 [Mus musculus] 42 F4 Cofilin-1 [Mus musculus] 74 F4

Tropomyosin alpha-4 chain [Mus musculus] 51 F4 14-3-3 protein zeta/delta [Mus musculus] 50 F4 Elongation factor 1-beta [Mus musculus] 49 F4 Ferritin heavy chain [Mus musculus] 48 F4 Peroxiredoxin-1 [Mus musculus] 166 F4

Histone H4 [Mus musculus] 65 F5 Ubiquitin [Mus musculus] 64 F5 Fatty acid-binding protein, epidermal [Mus musculus] 57 F5 Histone H2A type 1-F [Mus musculus] 37 F5 Profilin-1 [Mus musculus] 67 F5 Metallothionein-2 [Mus musculus] 63 F5 Fatty acid-binding protein, epidermal [Mus musculus] 132 F5 60S acidic ribosomal protein P2 [Mus musculus] 131 F5 Thioredoxin [Mus musculus] 72 F5 SH3 domain-binding glutamic acid-rich-like protein 3 [Mus musculus] 55 F5 Ubiquitin [Mus musculus] 43 F5 Fatty acid-binding protein, adipocyte [Mus musculus] 41 F5

Activated bone marrow macrophage

Ras GTPase-activating-like protein IQGAP1 [Mus musculus] 50 F1 Myosin-9 [Mus musculus] 57 F1 Myosin-11 [Mus musculus] 43 F1 Myosin-14 [Mus musculus] 43 F1 Talin-1 [Mus musculus] 60 F1 Filamin-A [Mus musculus] 111 F1

Heat shock protein HSP 90-alpha [Mus musculus] 87 F2 Integrin beta-2 [Mus musculus] 43 F2 Vacuolar protein sorting-associated protein 35 [Mus musculus] 46 F2

Endoplasmin [Mus musculus] 56 F2 Hexokinase-3 [Mus musculus] 55 F2 Coronin-7 OS=Mus musculus GN=Coro7 PE=2 SV=2 45 F2 Extended synaptotagmin-1 [Mus musculus] 42 F2

Moesin [Mus musculus] 89 F3 Vimentin [Mus musculus] 119 F3 Tubulin beta-5 chain [Mus musculus] 67 F3 Tubulin beta-2A chain [Mus musculus] 64 F3 Tubulin beta-3 chain [Mus musculus] 74 F3 Pyruvate kinase isozymes M1/M2 [Mus musculus] 207 F3 Heat shock protein 75 kDa, mitochondrial [Mus musculus] 55 F3 Heat shock cognate 71 kDa protein [Mus musculus] 87 F3 RNA-binding protein FUS [Mus musculus] 50 F3 V-type proton ATPase subunit B, brain isoform [Mus musculus] 49 F3 Transketolase [Mus musculus] 48 F3 Alpha-enolase [Mus musculus] 123 F3 Coiled-coil domain-containing protein 38 [Mus musculus] 41 F3 Gamma-enolase [Mus musculus] 44 F3 Lysosome-associated membrane glycoprotein 1 [Mus musculus] 37 F3 Galectin-3-binding protein [Mus musculus] 37 F3 Stress-70 protein, mitochondrial [Mus musculus] 49 F3 Sulfated glycoprotein 1 [Mus musculus] 78 F3 Heat shock protein HSP 90-beta [Mus musculus] 79 F3 Aldehyde dehydrogenase, mitochondrial [Mus musculus] 42 F3 ATP synthase subunit alpha, mitochondrial [Mus musculus] 46 F3 Plastin-2 [Mus musculus] 149 F3 Macrophage scavenger receptor types I and II [Mus musculus] 46 F3

78 kDa glucose-regulated protein [Mus musculus] 161 F3 Beta-enolase [Mus musculus] 98 F3

Peroxiredoxin-1 OS=Mus musculus GN=Prdx1 PE=1 SV=1 179 F4 Transgelin-2 [Mus musculus] 102 F4 Ras-related protein Rab-7a [Mus musculus] 113 F4 40S ribosomal protein S8 [Mus musculus] 38 F4 Galectin-3 [Mus musculus] 47 F4 Peroxiredoxin-4 [Mus musculus] 68 F4 ADP/ATP translocase 2 [Mus musculus] 58 F4 Annexin A5 [Mus musculus] 55 F4 Glyceraldehyde-3-phosphate dehydrogenase [Mus musculus] 120 F4 Annexin A4 [Mus musculus] 60 F4 Cathepsin B [Mus musculus] 66 F4 Cathepsin S [Mus musculus] 39 F4 Annexin A1 [Mus musculus] 128 F4 Macrophage-capping protein [Mus musculus] 84 F4 Fructose-bisphosphate aldolase A [Mus musculus] 45 F4 Twinfilin-1 [Mus musculus] 49 F4 Actin, cytoplasmic 1 [Mus musculus] 235 F4 Beta-actin-like protein 2 [Mus musculus] 105 F4 Synaptic vesicle membrane protein VAT-1 homolog [Mus musculus] 147 F4

10 kDa heat shock protein, mitochondrial [Mus musculus] 75 F5 Histone H4 OS=Mus musculus [Mus musculus] 81 F5 Parathymosin OS=Mus musculus [Mus musculus] 95 F5 Profilin-1 OS=Mus musculus [Mus musculus] 49 F5 Fatty acid-binding protein, epidermal [Mus musculus] 71 F5 Lysozyme C-2 OS=Mus musculus [Mus musculus] 74 F5

Ras-related C3 botulinum toxin substrate 2 [Mus musculus] 40 F5

Activated bone marrow macrophage + H. capsulatum Nonmuscle heavy chain myosin II-A [Mus musculus] 49 F1 Talin-1 [Mus musculus] 68 F1

Zinc transporter-4 OS=Mus musculus [Mus musculus] 82 F1 A disintegrin and metalloproteinase with thrombospondin motifs 57 F1 [Mus musculus] histone H4 (55AA) (1 is 3rd base in codon) [Mus musculus] 57 F1 Nonmuscle heavy chain myosin II-A [Mus musculus] 49 F1 Talin-1 [Mus musculus] 68 F1 Disintegrin and metalloproteinase domain-containing protein 17 [Mus 51 F1 musculus]

12-lipoxygenase [Mus musculus] 54 F2 Integrin beta-2 [Mus musculus] 38 F2

Signal transducer and activator of transcription 3 [Mus 62 F2 musculus] Ribonuclease Zc3h12a [Mus musculus] 77 F2 Disintegrin and metalloproteinase domain-containing protein 17 [Mus 51 F2 musculus] Zinc transporter 9 [Mus musculus] 65 F2 12-lipoxygenase [Mus musculus] 54 F2 Integrin beta-2 [Mus musculus] 37 F2 Polyubiquitin-C [Mus musculus] 59 F2 A disintegrin and metalloproteinase with thrombospondin motifs 57 F2 [Mus musculus]

Polyubiquitin-C [Mus musculus] 30 F3 ATP synthase subunit alpha, mitochondrial precursor [Mus musculus] 48 F3 pyruvate kinase M [Mus musculus] 111 F3

82 pyruvate kinase M [Mus musculus] 111 F3 Aldehyde dehydrogenase, mitochondrial [Mus musculus] 37 F3 Tubulin beta-5 chain [Mus musculus] 38 F3 Alpha-enolase [Mus musculus] 32 F3 serpin precursor [Mus musculus] 49 F3 Substance-P receptor [Mus musculus] 47 F3

Substance-P receptor [Mus musculus] 47 F4 cathepsin D precursor [Mus musculus] 67 F4 gamma actin-like protein [Mus musculus] 64 F4

A-X actin [Mus musculus] 64 F5 arginase-1 [Mus musculus] 52 F5 Metallothionein-2 [Mus musculus] 86 F5 ATP synthase subunit O, mitochondrial [Mus musculus] 34 F5 Cofilin-1 [Mus musculus] 30 F5 Histone H4 [Mus musculus] 81 F5 Histone H4 (55AA) (1 is 3rd base in codon) [Mus musculus] 57 F5

Supplementary Experimental Procedures

Sample preparation, and quantification by ICP-MS for total metal analysis

To perform total metal analysis, samples were digested by the wet acid method. In brief, in a 10

% nitric acid washed 2 ml vial, 100 l of cell lysates from macrophages were acidified with 50

l of concentrated trace metal grade nitric acid, 20 l of scandium (500 ppb) were added as internal standard to a final concentration of 10 ppb. The mixture was heated at 80 ºC for 3 h.

Then 50 l of hydrogen peroxide was added, heated at 80 ºC for 1 h and the final volume was made up to 1 ml. Three reagent blanks, and 3 SRMs were analyzed per sample batch.

The digested samples were quantified by external calibration method, using the following calibration points: 0, 0.1, 0.25, 0.5, 1, 2, 5, 10, 15 and 25 ppb; with Sc as internal standard at a concentration of 10 ppb. The collision reaction system was used to remove isobaric interference with 4 ml min-1 of He and a discrimination energy of 4 V. The forward power was 1550 watts, the dwell time 100 ms and the isotopes monitored were 24Mg, 34S, 44Ca, 45Sc, 55Mn, 56Fe, 57Fe,

63Cu, 66Zn, 68Zn, and 111Cd.

SEC-ICP-MS normalization of data

To normalize the response of ICP-MS signal from SEC separations on different days, 20 µl of 1 mg ml-1 solution of carbonic anhydrase was injected post-column to the LC system, and area of

Zn signals from samples was normalized to area of carbonic anhydrase peak. The absorbance at

280 nm of carbonic anhydrase was also followed to ensure integrity of the protein.

Protein in-solution tryptic digestion, proteomics and MASCOT search parameters

Fractions collected from SEC were concentrated by freeze drying. The pellet was re-suspended in 20 l of ammonium bicarbonate, denatured at 90 °C for 5 minutes and reduced with 3 l of 50 mM dithiothreitol (DTT) at 40 °C for 1 hr. Then alkylation of Cys residues was performed with

3 l of 50 mM iodoacetamide in dark at room temperature for 45 min. Three hundred nanograms of trypsin in 20 mM ammonium bicarbonate was then added and incubated overnight at 37 °C.

The reaction was stopped by adding 1 l of formic acid. The non-digested proteins and trypsin were removed by ultrafiltration using a 5 kDa MWCO filter, and filtrate was analyzed by nanoHPLC-ESI-MS-MS as described below.

Fractions from SEC-ICP-MS were prepared as described above for peptide mapping. An

Agilent 6300 series MSD Ion Trap XCT Ultra with a capillary binary pump was used for loading into microfluidic HPLC-Chip Zorbax SB 300A-C18 column and nano-flow binary pump to provide analytical flow for RP separation (Agilent Technologies). The ionization system utilized was a microfluidic chip, automatically loaded and positioned into the MS nanospray chamber.

Full scan mass spectra were acquired over m/z range 100–2200 in positive ion mode. For

MS/MS, experimental conditions were: m/z range: 100–2200; isolation width: 2 m/z units, fragmentation energy: 30–200%, maximum accumulation time: 40 ms.

To identify the proteins in each fraction, the results from LC-MS-MS were exported as

MASCOT generic file and submitted to the MASCOT MS-MS ion search engine, with the following parameters: database, SwissProt; taxonomy, mammals; enzyme, trypsin; missed cleavages, 1; variable modifications, carbamidomethylation of cysteine; peptide tolerance 1.6

Da; MS/MS tolerance 0.6 Da; peptide charge +1, +2, +3; instrument, ESI-TRAP.

Denaturation of MT fraction and treatment with Cd for SEC-ICP-MS analysis

A large volume of cell lysate (4 ml instead of 200 µl of usual experiments) from activated infected bone marrow macrophages was prepared by scaling up the described procedure. An aliquot was analyzed by SEC-ICP-MS to see the profile (Figure S1C), and the rest was concentrated to a volume of 1ml by using a MWCO filter (3kDa). This solution was injected

85 multiple times into the SEC column, and the fraction between 19 - 23 minutes was collected, freeze dried, re-suspended in ammonium acetate (50 mM), denatured by heat at 95°C for 5 minutes, and reduced by adding DTT to a final concentration of 10 mM. This solution was exposed to 5 mM CdCl2 for 30 minutes with stirring, followed by ultra-filtration using a 3kDa

MWCO filter and re-injected into the SEC-ICP-MS. Zn and Cd signals were monitored under standard instrumental conditions.

ICP-MS and SEC-ICP-MS quality control to avoid external Zn contamination

All total metal analysis experiments were carried out using trace metal grade reagents, on acid washed plastic vials and reagent blanks were used to correct the results. The analysis was done through a metal free auto sampler, rather than a HPLC flow injection method. The concentration of Zn in the blanks was always below 100 ppt, the blank estimate concentration on the calibration curves was always below 50 ppt, while the detection limits were below 30 ppt.

For chromatographic analysis, the mobile phase was cleaned using a Chelex 100 resin, using the batch method. In brief, an acid washed portion of 3 g of Chelex-100 was added to a liter of mobile phase, stirred for 30 min and passed through a 0.45 µm membrane. This decreased the Zn concentration below 200 ppt (measured as total). As an additional cleaning step a trapping column was packed with Chelex 100 resin, and placed just before the injector to avoid contamination from the HPLC pump. By doing this the base line for the ICP-MS Zn signal was below one thousand counts per second, which represents sub ppb levels.

Mice

Mice were housed in isolator cages in Department of Laboratory Animal Medicine, University of

Cincinnati, accredited by American Association for Accreditation of Laboratory Animal Care

(Frederick, MD).

Macrophage isolation, activation and H. capsulatum infection

Bone marrow from femurs and tibias of ~12 week old mice was cultured in RPMI

(BioWhittaker, MD) with 10% fetal bovine serum (HyClone Laboratories, Utah) and differentiated into macrophages with 10 ng/ml GM-CSF (Peprotech) for 7 days at 37°C and 5%

CO2. Cells were plated for 24 h with 10 ng/ml GM-CSF or TNF-α (Peprotech) before infection.

Peritoneal macrophages were isolated by peritoneal lavage using Hank‟s balanced salt solution

(HBSS), RBCs lysed, washed and rested for 24 h in media, after which they were treated for 24 h with 10 ng/ml GM-CSF or TNF-α before infection. Non-adherent cells were removed by washing, fresh media was added and macrophages were activated with cytokines at the time of infection.

Silencing

Silencing was performed by preparing an siRNA complex with TransIT TKO (Mirus Bio, LLC) in Opti-MEM Reduced Serum Medium (Life Technologies) followed by incubation for 30 min.

Macrophages were then transfected with the siRNA complex in antibiotic free RPMI media with

10% FBS and incubated for 12h followed by activation with GM-CSF for 24h. After this time, the media was replaced, GM-CSF added and macrophages were infected for 24h prior to analysis.

Gene expression analysis

RNA was extracted using RNeasy mini kit (Qiagen) or Trizol (Invitrogen, CA) and genomic

DNA was digested using RNase-free DNAse set (Qiagen) or DNase I recombinant, RNase free

(Roche). cDNA was prepared using Reverse Transcription Systems kit (Promega, WI). qRT-

PCR was performed in ABI Prism 7500, using Taqman assay with primer and probe sets from

Applied Biosystems, CA; hypoxanthine guanine phosphoribosyl transferase (Hprt) was used as

87 internal standard and target gene expression was normalized to resting peritoneal macrophages in peritoneal macrophage experiments and to activated bone marrow macrophages in bone marrow macrophage experiments.

SDS PAGE and western blotting

Cell lysates were prepared using Denaturing Cell Extraction Buffer (Invitrogen). To assess

STAT3 activation, macrophages were treated with GM-CSF and infected for 10 min, lysates were prepared and immunoblotted using antibodies for STAT3 (Millipore) and p-STAT3 Tyr705

(Cell Signaling). In silencing experiments, Stat3 and Stat5 were silenced, followed by activation and infection. Cell lysates were prepared 24 h post infection to assess p-STAT3 and p-STAT5

Tyr 694 (Cell Signaling). SDS-PAGE was performed on 10% Precise Protein Gel (Pierce). β- actin (Santa Cruz) was the loading control.

Confocal microscopy procedures, equipment and settings

Peritoneal and bone marrow macrophages were cultured on coverslips with cytokines. H. capsulatum was stained with PKH-26 (Sigma-Aldrich) as per manufacturer‟s protocol and were infected with PKH-26 stained yeasts. For microscopy, cells were washed twice with HBSS containing 0.5 mM ethylene diamine tetraacetate (EDTA) to chelate extracellular metals and stained for 30 min with nuclear stain, 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI)

(300 nM) and Golgi stain, BODIPY TR ceramide (Molecular Probes) (5 µM) and/or labile Zn stain, Zinpyr-1 (Santa Cruz Biotechnology) (10 µM) in RPMI only. Cells were washed and mounted on slides; images were acquired immediately on a Zeiss LSM710 confocal connected to

Zeiss Axio-observer.Z1 inverted microscope and visualized using ZEN 2011 software.

Macrophagecontrols: Bone marrow macrophages were plated on glass coverslips and treated with 2 µM ,N,N‟,N‟-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) overnight or 100 µM

ZnSO4 and 20 µM pyrithione (Sigma) for 30 min. Cells were washed twice with HBSS containing 0.5 mM EDTA and stained for 30 min with 10 µM Zinpyr-1. Cells were washed again and imaged as described above.

H. capsulatum control: Yeasts cultured in Ham‟s F12 were washed twice in HBSS and stained with PKH-26 dye as described in Methods. The yeasts were again washed twice in HBSS containing 0.5 mM EDTA and with stained with Zinpyr-1 and DAPI for 30 min. After two washes in HBSS, yeasts were imaged as described above.

All images were acquired as 8 bit images at room temperature using a Zeiss LSM 710, Zeiss

Axio observer.Z1 inverted microscope with 63X oil immersion/1.4 NA objective. Images were acquired with an optical slice thickness of 1 to 1.5 m and lateral pixel dimensions of 220 nm for regular images and 66 nm for zoom images. Acquisition settings were held constant for Zn fluorescence (green channel) in sample groups - Figures 3A-3E for all images; similar in Figure

3F and 3G for regular size images; and constant in Supplementary Figure 3 for all images.

Zinpyr-1 was excited using a 488 nm argon laser and emission was measured from 490 nm to

552 nm, DAPI was excited using a 405 nm violet laser diode and emission was measured from

410 to 487 nm and PKH-26 as well as BODIPY TR ceramide were excited using a 561 nm HeNe laser and emission was measured from 566 to 705 nm.

Specific depletion of Zn in macrophage culture media for ROS studies

Serum-free or serum-containing RPMI medium was mixed with 3g/L of Chelex-100 under sterile conditions. This solution was stirred overnight and filtered through a 0.45 µm filter. This treatment was repeated once more, and the resultant solution was analyzed to assess the percent of removal of the following elements: Zn, Cu, Cr, Mn, Co, and Fe. Then the levels of Cu, Cr,

Mn, Co and Fe were restored in RPMI (serum free and serum containing) using inorganic salts of

89 high purity of the corresponding element. The percent of Zn removal from serum-free and serum containing RPMI was 94% and 87 % respectively.

Growth inhibition assay

105 bone marrow macrophages were plated on a 96-well plate in culture media with or without

GM-CSF (Act-BM macrophage and BM macrophage respectively) for 24 h. The Act-BM macrophages were activated again with GM-CSF and both groups were infected with H. capsulatum at a multiplicity of 5 yeasts per macrophage. Growth of yeasts was assayed 24 h post infection. Macrophages were lysed hypotonically and yeasts plated on Mycosel agar 5 (Beckton

Dickinson) with 5% sheep blood and 5% glucose. Colonies were counted 7 days after incubation at 30°C; the detection limit was 100 colony forming units (CFU) and data expressed CFU X 106 per ml. Where H. capsulatum was cultured in ROS generating media, 200µM of xanthine substrate and 40mU or 60mU of XO was added to normal or low Zn media to generate ROS, with or without 10µM Zn. Enzyme and substrate solutions were prepared in Zn free solution or ddiH2O.

CHAPTER 3

IL-4 Regulates Zinc Homeostasis to Weaken Macrophage Defense Against an Intracellular

Pathogen

SUMMARY

The cytokine interleukin-4 (IL-4) dampens host immunity and resistance to infections. IL-4 weakens proinflammatory actions of macrophages by differentiating them into alternatively activated macrophages (M2 phenotype). We show that IL-4 dampens nutritional immunity by altering zinc (Zn) homeostasis in macrophages to favor growth of the intracellular fungus,

Histoplasma capsulatum. IL-4 treated macrophages increased labile Zn bioavailability in a time dependent manner, enhancing Zn uptake by the intracellular fungus. This was achieved by

STAT6-dependent elevation in the expression of metallothionein 3 (Mt3) and the Zn transporter,

Slc30a4 in macrophages. Silencing Mt3 and Slc30a4 attenuated the ability of IL-4 to increase labile Zn. Proteomic analysis identified cathepsins B, D and S in IL-4 stimulated infected macrophages; inhibition of cathepsins increased Zn bound to MT3, and subsequently decreased labile Zn. The intracellular yeasts manifested attenuated Zn acquisition in the absence of MT3 and upon inhibition of cathepsin activity. Based on these analyses we propose a mechanism by which IL-4 triggers Zn binding to MT3, followed by release of the metal upon action of cathepsins on MT3; a phenomenon that heightens Zn bioavailability, leading to increased acquisition of this metal by the intracellular fungus. These studies uncover the functional attributes of IL-4 that grant a non-protective and permissive phenotype to alternatively activated macrophages during fungal infection.

INTRODUCTION

Upon gaining entry into the host, several pathogens are phagocytosed by macrophages that form the first line of defense against infections. Activation in a proinflammatory environment curtails infection47, but depending on the cytokine milieu, these pathogens may replicate in macrophages to establish a niche within the host. The cytokine IL-4, has a profound role in tissue healing and wound repair96, but alternative activation of macrophages with this cytokine creates a favorable environment for survival of fungal pathogens and adversely affects host immunity against infections45, 57.

The production of IL-4 by eosinophils, basophils and epithelial cells stimulates differentiation of naïve T cells into the Th2 type, which in turn secrete more IL-4 giving rise to innate and adaptive responses that poorly combat infection97. In murine models of fungal infections, an excess of IL-4 is associated with elevated pathogen load caused by a weakened proinflammatory response48, 53. Exposure of macrophages to IL-4 induces differentiation into the

M2 phenotype, characterized by enhanced expression of markers such as arginase-1, Ym1, Fizz1

(found in inflammatory zone 1) and mannose receptor (CD206)70. These factors are representative of a shift from classically activated to an alternative macrophage phenotype.

The availability of metals is indispensible for survival of pathogens as they support numerous biological processes and are essential for pathogen defense functions. Thus, redistribution of host elements such as Zn, Fe and Cu during pathogen invasion is a critical step in determining resistance to infection. Interferon-γ (IFN-γ) decreases Fe acquisition by decreasing transferrin receptor and enhancing ferroportin-1 expression58. We showed that the pro-inflammatory cytokine GM-CSF imposes nutrient deprivation by restricting Zn access to the fungal pathogen H. capsulatum, and concomitantly enhances superoxide burst, thereby

93 strengthening the antifungal defense in macrophages. Therefore, innate immune stimuli that foster a pro-inflammatory environment can restrict basic metals essential to pathogen survival. A contrasting phenomenon deployed by a permissive, weakened immune response may be viewed as alteration of metal homeostasis which culminates in favorable metal acquisition, improved pathogen defenses and survival. In GM-CSF activated macrophages infected with H. capsulatum, IL-4 presents a survival advantage to the fungus, in that, the growth of yeasts is enhanced by this cytokine45. IL-4 triggers increased expression of transferrin receptor (TfR) in uninfected macrophages, suggesting improved Fe uptake. However, this effect is not observed during infection48, suggesting that increased survival of yeasts may not have resulted from improved Fe availability. In this regard, the concept that functional attributes of alternative activation may be governed by modulation of metal homeostasis has not been visited.

Metallothioneins (MTs) are a class of metal binding proteins that regulate physiological

Zn homeostasis in cells and regulate availability of labile Zn. Mt1 and Mt2 manifest a ubiquitous expression pattern26 and in agreement with this, macrophages express high levels of Mt1 and Mt2 and these MTs bind and sequester Zn. The expression Mt3 however, has been chiefly associated with expression in the brain and may be involved in Zn binding followed by subsequent release of the metal98. The spatial dynamics of Zn acquisition or Zn release and the stimuli that trigger these events have not been thoroughly investigated. Exposure to oxidative stress causes Zn release from MT3 in astrocytes. Cathepsins degrade MTs in lysosomes99, however whether degradation of MTs stimulates release of the metal or vice versa is unclear. Zn transporters also facilitate Zn mobilization in the cell and can contribute to an increase in Zn availability in organelles. The Zn exporter, ZNT4 locates on endosomal membranes and exports cytosolic Zn

94 into this organelle23. As endosomes fuse with lysosomes, ZNT4 may contribute to an increase in

Zn within phagolysosomes of infected macrophages.

We have previously demonstrated that Zn is critical for survival of H. capsulatum, and restriction of this metal sharply attenuates fungal growth45. In this work, we investigated the mechanism by which alternative activation of macrophages with IL-4 targets Zn modulation to abate host defenses and create a permissive environment favoring pathogen survival. We elucidate the pathway by which IL-4 regulates Mt3 and Slc30a4, leading to time-dependent increase in the availability of labile Zn in macrophages, culminating in augmented acquisition of

Zn by the intracellular fungal pathogen.

RESULTS

IL-4 elevates labile Zn in macrophages and enhances acquisition by H. capsulatum

We investigated modulation of Zn in IL-4 treated bone marrow-derived macrophages infected with H. capsulatum. Inductively coupled plasma mass spectrometric analysis in conjunction with size exclusion chromatography (SEC-ICP-MS) was used to examine Zn distribution across different molecular masses in macrophage lysates. IL-4 triggered an increase in the Zn signal occurring in the SEC fraction at ~26 min (Figure 1A). The species eluting at this time point correlate with a molecular weight of <1.5kD, based on the standards indicating the presence of

„labile‟ or exchangeable Zn in this fraction. In contrast to GM-CSF activated macrophages, IL-4 did not dramatically increase Zn binding to the MT1 and MT2 peak at ~20 min, but did show appearance of a Zn binding peak at ~23 min; this peak was also observed in untreated macrophages (Figure 1A). This fraction showed UV-Vis spectral absorbance maxima at 260 nm, characteristic of metallothioneins which lack aromatic amino acid residues (data not shown). In this text, we refer to the peak as the „MT3 signal‟. The percent contribution of labile Zn as calculated by peak area from 24.5 min to 30 min in IL-4 treated-infected macrophages was 19%, as opposed to 11.5% in untreated cells and 3% in GM-CSF activated macrophages (Figure 1B).

Next, we examined total Zn in yeasts isolated from IL-4 treated macrophages. In correlation with an increase in macrophage labile Zn, we found that yeasts from IL-4 treated macrophages amassed more Zn compared to untreated controls (Figure 1C). These data indicate a role for IL-

4 in mediating Zn redistribution to augment the availability of labile Zn.

IL-4 alters Mt and Zn transporter expression in macrophages

To understand the phenomenon of Zn modulation in IL-4 treated macrophages, we analyzed expression of MTs and Zn transporters. IL-4 treated uninfected and infected cells exhibited >9 fold increase in Mt3 expression over untreated macrophages. The expression of Mt1 and Mt2 were only modestly altered (Figure 2A). Next, we asked whether the changes in labile Zn observed upon IL-4 exposure resulted from altered Zn transport. We determined the expression of 14 Zn importers, Slc39a (ZIPs) and 10 Zn exporters, Slc30a (ZNTs) and found that IL-4 induced the expression of Slc30a4 in both uninfected and infected bone marrow macrophages compared to untreated macrophages, and a ~5 fold increase in Slc39a2 in IL-4 treated-infected cells was observed (Figures 2B and 2C). To determine whether the alteration of Zn homeostasis occurred immediately upon IL-4 exposure, we investigated Mt3 and Slc30a4 expression at 15 min, 30 min, 1 h and 3 h in macrophages. The elevation in Mt3 and Slc30a4 expression was seen as early as 15 min upon treatment with IL-4 (Figures 2D and 2E). These results suggest that IL-

4 may trigger rapid changes in Zn availability post infection. The modest increase in Slc39a2 indicates that IL-4 induces Zn import, albeit not to the same extent as GM-CSF and enhanced

Slc30a4 and Mt3 expression may facilitate redistribution of labile Zn in IL-4 treated macrophages to favor H. capsulatum survival.

The promoter region of Mt genes contains consensus STAT binding sites30, but a role for

STAT proteins in the regulation of Mt3 has not been demonstrated. IL-4 signals via STAT6 and the immediate response of Mt and Zn transporter expression suggested that these were directly regulated by STAT6. Indeed, Stat6-/- macrophages failed to upregulate Mt3 and Slc30a4 expression at 24 h post infection, suggesting a role for STAT6 signaling in regulating Zn homeostasis (Figures 2F and 2G). Moreover, the upregulation of Mt3 and Slc30a4 at 15 min

97 and 30 min in response to IL-4 was impaired in Stat6-/- macrophages, indicating that STAT6 directly regulated Mt3 and Slc30a4 expression (Figures 2H and 2I). In addition, this effect was specific to Stat6 as Stat3 silenced macrophages responded normally to IL-4 stimulated increase in Mt3 (Figure 2J).

MT3 and ZNT4 increase labile Zn in macrophages

To establish the functional significance of MT3 in favoring fungal survival, we silenced the gene by RNA interference and analyzed Zn distribution in macrophages. Silencing Mt3 led to ~80-

93% downregulation in the expression of this gene (Figure 3A). We observed a reduction in the labile Zn peak at ~26 min of the SEC chromatogram in IL-4 treated and infected macrophages upon Mt3 silencing compared to scramble treated control. In addition, a decrease in the Zn signal was observed at ~23 min, suggesting that this peak contained MT3 (Figure 3C). Next, we examined the effect of ZNT4 in contributing to the increase in labile Zn. Slc30a4 silencing led to

~95% reduction in expression in IL-4 stimulated and infected cells (Figure 3B). Silencing

Slc30a4, like Mt3 led to a partial decrease in the labile Zn fraction in IL-4 treated macrophages.

Combined silencing of both, Mt3 and Slc30a4 produced a greater, pronounced reduction in labile

Zn (Figure 3C). These results indicate a role for Mt3 and Slc30a4 in altering Zn availability under the influence of IL-4 in macrophages. The labile Zn fraction, albeit small, is a readily accessible Zn source. Our previous results (Chapter 2) and current observations justify a direct correlation between labile Zn and Zn acquired by the intracellular pathogen. Therefore, we investigated whether the decrease in labile Zn resulting from Mt3 silencing abated fungal Zn acquisition. Yeasts obtained from Mt3 silenced IL-4 treated macrophages manifested decreased

Zn acquisition (Figure 3D) compared to yeasts recovered from scramble treated control,

98 suggesting a role for MT3 in mediating free Zn release and acquirement by the fungus. We speculate that the unique properties of MT3, distinct from that of MT1 and MT2 (as indicated in the discussion section) are responsible for labile Zn release by the protein.

Cathepsins regulate Zn acquisition by H. capsulatum

Alternative activation of macrophages with IL-4 enhances proteolytic activity by cysteine cathepsins, CtsB and CtsS100. The release of Zn and its acquisition by H. capsulatum within phagosomes of IL-4 treated macrophages was partially dependent on Mt3. Though the expression of Mt3 increased by >9 fold in IL-4 treated cells, it corresponded to a relatively small distinct Zn binding peak at 23 min, predicted as the MT3-Zn signal in this study. We therefore postulated that MT3 was being synthesized and degraded by induction of cathepsin activity in macrophages. We probed the involvement of cathepsins using broad cathepsin inhibitors

Pepstatin A (PepA) and N-Acetyl-Leu-Leu-Met-CHO (ALLM). Treatment of macrophages with

PepA and ALLM led to a 5.3% increase in the Zn signal associated with the MT3 peak (Figure

4A), and a concomitant 7.5% decrease in the labile Zn fraction (Figure 4B) in H. capsulatum infected IL-4 treated macrophages, suggesting that cathepsin activity was involved in degradation of Zn bound MT3 and the release of Zn in infected macrophages. The cause of increased labile Zn in uninfected IL-4-treated cells upon cathepsin inhibition is unclear. To analyze the impact of cathepsin inhibition on Zn acquisition by the fungus, we analyzed total Zn in yeasts obtained from IL-4 stimulated macrophages that were left untreated or treated with the inhibitors. There was a 30% decrease in the concentration of Zn, but not Fe in yeasts recovered from cathepsin inhibited macrophages (Figures 4C and 4D). These data indicate that increased proteolytic activity in IL-4 treated macrophages leads to degradation of Zn binding proteins

99 including MT3, mediating the release of free Zn, which results in increased bioavailability of the metal for uptake by the fungal pathogen.

To evaluate the dynamics of labile Zn increase, and to further support our findings for the role of cathepsin driven proteolysis in Zn elevation, we analyzed changes in labile Zn in IL-4 treated and infected macrophages over time. The intensity of labile Zn signal showed an 18.9% increase in IL-4 treated macrophages and an even greater increase (54.7%) in IL-4 treated and infected macrophages at the 24h time point, compared to 0h and 1h respectively (Figure 4E), indicating that IL-4 triggered a gradual increase in the exchangeable fraction of Zn over time.

Collectively, the data emphasize that IL-4 augments fungal Zn acquisition by enhancing labile

Zn availability in macrophages.

100

DISCUSSION

This study elucidates a potential mechanism by which modulation of Zn homeostasis impacts the functional attributes of alternatively activated macrophages (Figure 5). The characteristics of IL-

4-differentiated M2 macrophages that distinguish them from protective „classically activated‟

M1 macrophages have been vastly investigated101. We introduce the concept that IL-4 triggered changes in labile Zn may form a characteristic feature of the permissive phenotype of M2 macrophages. Our studies reveal a mechanism by which heightened release of labile Zn enhances permissiveness to fungal growth in IL-4 treated macrophages.

Exposure of GM-CSF differentiated macrophages to IL-4 induced a change in Zn distribution which was distinct from that of GM-CSF activation, suggesting that a shift in macrophage phenotype may associate with a change in Zn homeostasis. IL-4 treated macrophages „expanded‟ their labile Zn compartment and therefore harbored a greater amount of accessible Zn. In infected macrophages, the amount of exchangeable Zn directly influences the ability of intracellular yeasts to secure the metal from host resources (Chapter 2). By increasing

Zn availability, the environment for Zn acquisition by the fungus was made more conducive. In agreement with this, H. capsulatum yeasts had amassed a greater concentration of Zn in IL-4 treated macrophages. Of note, the increase in labile Zn was characteristic of IL-4 signaling. GM-

CSF triggers Zn deprivation, an effect opposite to that of IL-4, and TNF-α fails to induce any change in Zn proteome distribution of macrophages (Chapter 2). Whether the alternatively activating cytokine, IL-13, which shares the IL-4Rα chain with IL-4102, also elevates labile Zn in macrophages, remains to be determined. The labile Zn increase by IL-4 is distinct from Zn modulation observed in a classically activated macrophage phenotype, and may be a unique property of alternative activation.

101

The role of MTs in regulating physiological Zn levels in cells is well established103.

However, the mechanism by which Zn exchange by MTs influences macrophage defenses is not understood. MT3 was initially identified as a neuronal growth inhibitor factor (GIF), and has been largely regarded as a brain specific isoform of MT98. In general, transcriptional induction of

MTs is dependent on metal responsive transcription factor 1 (MTF-1)29, but the unique expression pattern of Mt3 versus ubiquitous expression of Mt1 and Mt2 suggests the involvement of other mediators in regulation of Mt gene expression. MTF-1 may form complexes with additional co-factors to direct expression of different Mt genes104. We found that Mt3 expression in macrophages was specifically controlled by STAT6 signaling. While a role of MTF-1 has not been eliminated, action of this transcription factor in immune cells may very well be influenced by activated STAT molecules via direct interactions.

It is broadly accepted that MTs bind and sequester Zn with picomolar affinity105, but the

Zn binding affinity of MT3 has not been specifically defined. MT3 may exhibit Zn binding and donating properties distinct from other MT counterparts, in that, MT3, but not MT1 and MT2 has been related to „Zn release‟ in neuronal models of oxidative stress98 and in Alzheimer‟s disease106. Although the protein shows 70% similarity to other members of the family, MT3 possesses two unique structural properties that set it apart from other MTs, a TCPCP motif, and an additional hexapeptide insertion away from the metal binding cluster. These differences impart an „unstable‟ and open conformation that alters the dynamic interaction of the Zn-thiolate cluster of MT3 with the surrounding environment107. In agreement with this, Mt3 upregulation in

IL-4 treated macrophages contributed to the increase in labile Zn. Whether MT3 releases inherently bound Zn or enhances Zn mobilization from storage organelles or acquires the metal from other proteins, subsequently releasing it upon IL-4 exposure is not known. Based on our

102 analysis, if untreated cells harbored Zn in labile form in storage organelles, this fraction would be evident in the SEC-ICP-MS chromatogram. However, the labile fraction increased post IL-4 stimulation, suggesting that the source of exchangeable Zn is imported or acquired from other proteins by MT3 prior to release.

IL-4 stimulated an alteration in Zn transport by enhancing Slc39a2 and Slc30a4 expression in macrophages. While ZIP2 has largely been ascribed as a plasma membrane Zn transporter108, the spatial distribution of ZNT4 may vary depending on specific stimuli. ZNT4 associates with the Golgi in mammary epithelial cells37, while other studies have identified this transporter on the endosomal membrane22. This indicates that the spatial localization of ZNT4 may be governed by molecular cues that regulate Zn redistribution in macrophages. We postulate that in IL-4 treated infected macrophages, ZNT4 eventually localizes on the phagolysosomal membrane derived by endosome-lysosome fusion with phagosomes. Thus, cytosolic Zn may be driven into the phagosomes via ZNT4, thereby elevating labile Zn within this compartment and resulting in increased Zn acquisition by yeasts in the phagosomes. Our results indicate that IL-4 shapes Zn homeostasis by modulating MTs and transporters in a manner resulting in elevated Zn acquisition by the intracellular pathogen.

Our investigation of dynamics of labile Zn increase over time and the role of cathepsins indicates that Zn release by MT3 may be under the control of proteolytic activity in phagolysosomes. Inhibition of cathepsins resulted in an increased Zn-bound MT3 signal, and a concomitant decrease in labile Zn in infected macrophages, suggesting that MT3 is degraded by cathepsins to mediate Zn release. It can be speculated that MT3 is transported to the phagolysosomes prior to degradation. The role of cathepsins in driving Zn release is further supported by the reduction in the ability of intracellular yeasts to amass Zn. It should be noted

103 that the inhibitors of cathepsins act on other proteases and the observed effects may have resulted from a broader proteolytic action. It is also possible that Zn acquisition by H. capsulatum is partly facilitated by secretion of fungal proteases that degrade host proteins for Zn acquisition.

Though cathepsin-like proteins have not been identified in fungi, some fungal peptidases may have structural homology to cathepsins109, which can function similarly in proteolytic degradation of host proteins.

IL-4 reverses the growth-inhibitory action of GM-CSF and favors H. capsulatum survival in macrophages45. Our studies in Chapter 2 have established that Zn promotes growth and the deficiency of this metal is detrimental to fungal survival. Additional studies are necessary to thoroughly dissect the molecular mechanisms that direct IL-4 driven Zn changes. Whether the loss of H. capsulatum associated Zn in the absence of MT3 attenuates fungal survival needs to be assessed. This will confirm the significance of MT3 as a function of IL-4 in shaping an environment suitable for pathogen growth. The impact of ZNT4 on Zn acquisition and survival of the intracellular pathogen needs to be demonstrated. We speculate that both, MT3 and ZNT4 contribute to augmented Zn acquisition by the intracellular pathogen. If this is true, combined silencing of Mt3 and Slc30a4 will result in marked decrease in fungal Zn acquisition leading to inhibition of fungal growth. Additionally, because STAT6 regulates expression of both Mt3 and

Slc30a4, the direct contribution of STAT6 in enhancing labile Zn must be analyzed.

The functional properties of alternatively activated macrophages in the context of infectious immunity have been widely investigated; however a role for Zn homeostasis has not been defined. Through this study, we propose that Zn modulation increases the permissiveness of

IL-4 stimulated macrophages. We present evidence that the brain specific isoform of MT, Mt3 is expressed in murine macrophages and is regulated by STAT6 signaling. By orchestrating the

104 regulation of MT3 and Zn transporters, IL-4 skewed the balance in Zn distribution by increasing labile Zn, which facilitated greater Zn acquisition by the intracellular pathogen. In summary, these data present a conceptual advance into the emerging roles of IL-4 in shaping alternative activation of macrophages, leading to diminished protective immunity against infections.

EXPERIMENTAL PROCEDURES

Mice

C57BL/6 WT mice were obtained from Jackson Laboratory and Stat6-/- mice were kindly provided by Dr. Senad Divanovic (Cincinnati Children‟s Hospital Medical Center). Mice were housed in isolator cages in the Department of Laboratory Animal Medicine, University of

Cincinnati, accredited by American Association for Accreditation of Laboratory Animal Care

(Frederick, MD). All animal experiments were in accordance with Animal Welfare Act guidelines of National Institutes of Health.

Macrophage isolation, IL-4 stimulation and H. capsulatum infection

Bone marrow from femurs and tibias of ~12 week old mice was cultured in RPMI

(BioWhittaker, MD) with 10% fetal bovine serum (HyClone Laboratories, Utah) and differentiated into bone marrow macrophageswith 10 ng/ml GM-CSF (Peprotech) for 7 days at

37°C and 5% CO2. Macrophages were plated for 24 h with 10 ng/ml IL-4 (Peprotech) or left untreated before infection. After 24h, media was changed and macrophages were stimulated with

10 ng/ml IL-4 prior to infection. For infection, strain G217B was cultured in Ham‟s F12 (Zn concentration, 250 ± 18 ng/ml or 4 µM) and washed twice with Hank‟s balanced salt solution

(HBSS) and infected at a dose of 5 H. capsulatum yeasts per macrophage.

Silencing

105

Genes were silenced in macrophages using TransIT-TKO transfection reagent (Mirus Bio LLC) and 100nM Mt3 (Santa Cruz), Slc30a4, or scrambled siRNA (Dharmacon) as per manufacturer‟s instructions. Briefly, the siRNA was complexed with TransIT TKO in Opti-MEM Reduced

Serum Medium (Life Technologies) followed by incubation for 30 min. acrophages were then transfected with the siRNA complex in antibiotic free RPMI media with 10% FBS and incubated for 12h followed by stimulation with IL-4 for 24h. After this time, the media was replaced, IL-4 added and cells were infected with H. capsulatum for 24h prior to analysis.

RNA extraction and gene expression analysis

RNA was extracted using RNeasy mini kit (Qiagen) or Trizol (Invitrogen, CA) and genomic

DNA was digested using RNase-free DNAse set (Qiagen) or DNase I recombinant, RNase free

(Roche) at the indicated time points, or at the end of 24h. cDNA was prepared using Reverse

Transcription Systems kit (Promega, WI). qRT-PCR was performed in ABI Prism 7500, using

Taqman assay with primer/probe sets from Applied Biosystems, CA; hypoxanthine guanine phosphoribosyl transferase (HPRT) was used as internal standard and target gene expression was normalized to untreated macrophages.

Cathepsin inhibition

Macrophages were treated with DMSO control or with the inhibitors PepA (1µM), ALLM

(1µM) or both for 3h, followed by overnight stimulation with IL-4. After this time, media was changed, inhibitors and IL-4 were added, followed by infection with yeasts for 24h prior to SEC-

ICP-MS analysis.

Cell lysates for ICP-MS

Cells were washed and lysed with 0.1% sodium dodecyl sulfate (SDS) in HPLC water for 20 min on ice. For time point analysis, macrophages were lysed at 1h, 9h and 12h intervals. Lysates

106 were passed through a 0.22 µm filter or centrifuged at 13,000 rpm for 5 min for lysate analysis.

For total metal analysis of H. capsulatum, lysates were centrifuged at 13,000 rpm for 5 min to pellet the yeasts and the pellet was washed twice with HBSS.

Total elemental analysis and SEC-ICP-MS screening

ICP-MS analysis was performed on Agilent 7700x ICP-MS instrument (Agilent Technologies).

A conventional Meinhard nebulizer, Peltier-cooled spray chamber, and shield torch constituted the sample introduction system under standard conditions. Sc was used as internal standard and

SRM (DORM-3 or NIST1745) were analyzed for every digestion. For SEC-ICP-MS, Agilent

1100 series HPLC system equipped with a binary pump, vacuum membrane degasser, thermostated auto sampler, column oven, and diode array detector (DAD), with a semi-micro flow UV-Vis cell was coupled to the ICP-MS through a 0.17mm internal diameter short PEEK tube. The system was controlled using Chemstation software. The column used was TSK Gel

3000SW 7.5 x 300 mm was used.

Statistics p-values were calculated using non-paired Student t test where two groups were compared;

*p<0.05; **p<0.01; NS, not significant.

107

FIGURE LEGENDS:

Figure 1: IL-4 increases macrophage labile Zn and acquisition by H. capsulatum

(A) SEC-ICP-MS chromatogram of untreated, IL-4 treated and GM-CSF activated macrophages infected with H. capsulatum (Hc), y axis represents offset Zn signal, zoom in of labile Zn fraction on the right; (B) Concentration of labile Zn calculated by peak area between 24 min and

30 min, error bars are mean ± SD from two independent experiments; (C) Total Zn in H. capsulatum yeasts recovered from untreated and IL-4 treated macrophages, error bars are mean ±

SD from 2 independent experiments.

Figure 2: IL-4 upregulates the expression of Mt3 and Slc30a4 via STAT6

Gene expression analysis of (A) Mt1, Mt2 and Mt3; (B) Slc39a1 – Slc39a14 and (C) Slc30a1-

10 in untreated, IL-4 treated and infected bone marrow (BM) macrophages, normalized to untreated control; Hc, H. capsulatum; ND, not detected, data are from 2 independent experiments; (D and E) Time point analysis of Mt3 and Slc30a4 expression in IL-4 treated uninfected and infected macrophages at 15 min, 30 min, 1h and 3h, normalized to expression in untreated cells at 0 min time point, data are from 1 experiment; (F and G) Expression of Mt3 and Slc30a4 in untreated infected macrophages and IL-4 treated uninfected and infected macrophages obtained from Stat6-/- mice normalized to untreated control from 2 independent experiments; (H and I) Time point analysis of Mt3 and Slc30a4 expression in IL-4 treated uninfected and infected WT and Stat6-/- macrophages at 15 min and 30 min, normalized to Mt3 and Slc30a4 expression in WT macrophages at 0 min time point, data are from 1 experiment; (J)

Expression of Mt3 in scramble siRNA or Stat3 siRNA treated macrophages stimulated with IL-4 and infected, normalized to scramble siRNA treated cells; error bars are mean ± SEM.

108

Figure 3: Mt3 and Slc30a4 mediate labile Zn increase by IL-4

(A and B) Gene expression analysis in Mt3 and Slc30a4 silenced cells compared to scramble siRNA treated macrophages, percent values represent decrease over respective scramble groups, two independent experiments, error bars are mean ± SEM; (C) SEC-ICP-MS analysis of Mt3 and

Slc30a4 silenced IL-4 treated and infected macrophages compared to scramble siRNA treated control; (D) Total Zn in H. capsulatum yeasts recovered from scramble siRNA and Mt3 siRNA treated macrophages stimulated with IL-4 and infected with H. capsulatum.

Figure 4: Cathepsins regulate MT3 mediated Zn delivery to intracellular yeasts

(A) Zn signal related to MT3 peak from 22.7 min - 24 min, and (B) the labile Zn peak from 24.3 min to 30 min of the SEC-ICP-MS chromatogram calculated as peak area in IL-4 stimulated macrophages left untreated or treated with cathepsin inhibitors, CPS, counts per second, data are from 1 experiment; (C and D) Total Zn and Fe in yeasts recovered from IL-4 stimulated macrophages with or without cathepsin inhibitors, data are from 2 independent experiments, error bars are mean ± SD; (E) Time point analysis showing increase in labile Zn signal from 0h to 24h in IL-4 treated and infected macrophages, BM, bone marrow, Hc, H. capsulatum, representative of 2 independent experiments.

Figure 5: Schematic illustration of Zn regulation by IL-4

(1) Entry of H. capsulatum yeasts into macrophages by phagocytosis and formation of the phagolysosome; (2) IL-4 binds IL-4 receptor complex and activates Jak2-STAT6 signaling; (3)

Activated STAT6 enters the nucleus and drives transcription of STAT6 responsive genes; (4)

STAT6 dependent expression of Mt3, and binding of MT3 to Zn; (5) STAT6 dependent

109 expression of Slc30a4, and export of cytosolic Zn into the phagolysosome; (6) Induction of an additional Zn binding member, which may interact with ZNT4 and donate Zn for transport into the phagolysosome; (7) Zn bound MT3 migrates to the phagolysosome where cathepsins recognize the protein; (8) Degradation of MT3 by cathepsins S, B and D mediates release of bound Zn; (9) Zn inhibits proton flux via HV1 channel; (10) Inhibition of HV1 function dysregulates charge compensation and inhibits ROS generation via NADPH oxidase (Nox); (11)

Labile Zn released from MT3 and imported by ZNT4 collectively enhances permissiveness and facilitates survival of the fungal pathogen; solid lines, established links in this study and/or in literature; dotted lines, predicted links.

110

FIGURES:

Figure 1:

111

Figure 2:

112

113

Figure 3:

114

Figure 4:

115

Figure 5:

116

CHAPTER 4

Selectivity and Specificity of Small Molecule Fluorescent Dyes/Probes Used For The

Detection of Zn2+ And Ca2+ In Cells£

&Julio A. Landero-Figueroa1, &Kavitha Subramanian Vignesh2, George Deepe3,4, and

Joseph Caruso1*

1Metallomics Research Center, Department of Chemistry, McMicken College of Arts and

Sciences, University of Cincinnati, Cincinnati, OH 45221, USA

2Department of Molecular Genetics, Biochemistry, Microbiology and Immunology, University of Cincinnati, Cincinnati, OH 45267, USA

3Division of Infectious Diseases, College of Medicine, University of Cincinnati, Cincinnati, OH

45267 USA.

4Veterans Affairs Hospital, Cincinnati, OH 45220, USA.

*corresponding author, email: [email protected]

&JALF and KSV provided equal contributions to this study.

£Submitted to Metallomics, Royal Society of Chemistry Publishing, Cambridge UK, October

2013

117

ABSTRACT

Fluorescent dyes are widely used in the detection of labile (free or exchangeable) Zn2+ and Ca2+ in living cells. However, their specificity over other cations and selectivity for detection of labile vs. protein-bound metal in cells remains unclear. We characterized these important properties for commonly used Zn2+ and Ca2+ dyes in a cellular environment. By tracing the fluorescence emission signal along with UV-Vis and size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) in tandem, we demonstrated that among the dyes used for Zn2+, Zinpyr-1 fluoresces in the low molecular mass (LMM) region containing labile Zn2+, but FluoZinTM-3 AM, Newport GreenTM DCF and Zinquin ethyl ester display weak fluorescence, lack of metal specificity and respond strongly in the high molecular mass (HMM) region. Four

Ca2+ dyes were studied in an unperturbed cellular environment, and two of these were tested for binding behavior under an intracellular Ca2+ release stimulus. A majority of Ca2+ was in the labile form as tested by SEC-ICP-MS, but the fluorescence from Calcium Green-1TM AM, Oregon

Green® 488 BAPTA-1, Fura redTM AM and Fluo-4 NW dyes in cells did not correspond to free

Ca2+ detection. Instead, the dyes showed non-specific fluorescence in the mid- and high- molecular mass regions containing Zn, Fe and Cu. Proteomic analysis of one of the commonly seen fluorescing regions showed the possibility for some dyes to recognize Zn and Cu bound to metallothionein-2. Our studies indicate that Zn2+ and Ca2+ binding dyes manifest fluorescence responses that are not unique to recognition of labile metals and bind other metals, leading to suboptimal specificity and selectivity.

118

INTRODUCTION

Metal homeostasis is tightly regulated in living cells. Numerous biological stimuli such as hormones, pathogens and tumorigenesis trigger responses that alter the total and labile metal fraction in cells and their distribution within intracellular organelles110-114. Modulation of metals controls important cellular processes including transcription, enzyme function, signaling, and metabolism110, 115-119. Thus, analysis of metal flux provides mechanistic insights into cell function.

Atomic absorption spectroscopy (AA), inductively coupled plasma emission spectrometry (ICPAES) and inductively coupled plasma mass spectrometry (ICP-MS) have been invaluable in accurate metal detection and quantification in biological samples66. But, they do not allow visualization of metal flux and distribution within cells. The development of small molecule, cell permeable fluorescent dyes has facilitated detection of metal flux and distribution in real time on living cells through confocal microscopy120. These dyes fluoresce once coordinated with the metal ions or metal ion species that are different chemical forms of the same metal. Modification with acetoxy-methyl (AM) esters for permeability into cells has enhanced the applicability of these dyes121. The combined use of red and green fluorescent dyes enables simultaneous imaging of two metals in cells122. These advantages and others such as ratiometric measurement123 and quantification of fluorescent signals124 have led to an increasingly popular use of metal binding dyes in microscopy and flow cytometry. Since their initial development, many dyes have been generated with specificity to metals such as Ca, Zn and Fe.

Zn is the second most abundant transition metal in living cells that is essential for cellular processes such as transcription, translation, enzyme function, protein folding and signaling125-127.

119

Cells possess a labile Zn2+ pool that may be stored in organelles such as the Golgi apparatus, endoplasmic reticulum (ER) and zincosomes36, 128, 129. Imaging Zn2+ using Zinpyr-1TM,

FluoZinTM-3 AM or Newport GreenTM DCF dyes has contributed to the current understanding of intracellular flux and distribution of Zn in cells such as those of the immune system, pancreatic islets and neurons13, 130-132. Intracellular Ca2+ imaging has been extensively employed in studying mobilization, release and uptake by mitochondria and ER in cells such as cardiomyocytes and neurons133, 134. Widely used probes for Ca2+ include Calcium Green-1TM AM, Fura-2 AM, Fura redTM AM, Fluo-3 AM, Fluo-4 and Oregon Green® 488 BAPTA-1 AM133, 135-138.

Specificity and sensitivity of fluorescent dyes is influenced by intracellular factors including pH, concentration of competing metals, ionic strength, preference for hydrophobic regions and binding constants85, 120, 139, 140. Commercially available dyes have been characterized for these parameters, largely in buffered solutions, but their behavior within the complex intracellular environment has remained unclear141, 142. While suitable for partial characterization, these solutions lack cellular constituents such as membranes, organelles and transporters that are required to bind, store or transport metals. Binding properties of dyes in buffered solutions may be quite different from their behavior in an intracellular environment. For example, cellular pH varies spatially and temporally from an acidic pH of 5.4 to a physiological pH of 7.4143, 144. The changes in pH may cause differential behavior by dyes in different compartments, where binding site availability is typically a function of pH. The fluorescence signal is influenced by the strength of these interactions, and quantification of labile or exchangeable metal pools within cells may be challenging. Given the extensive use of metal binding dyes in cellular imaging and flow cytometry to obtain data about relative metal concentration, exchangeable pools and

120 trafficking, additional studies are necessary to dissect their specificity to particular metals and selectivity for metal binding species within the complex cellular environment.

In this study, we have defined specificity to mean binding to a particular metal ion, while selectivity refers to one or more molecular weight regions in which binding to a specific metal ion takes place. We characterized the specificity and selectivity of four Zn2+ binding and four

Ca2+ binding dyes in an intracellular environment using a bone marrow macrophage cell line.

The Zn2+ dyes selected were Zinpyr-1, FluoZinTM-3 AM, Newport GreenTM DCF and Zinquin ethyl ester, and those chosen for Ca2+ were Calcium Green-1TM AM, Oregon Green® 488

BAPTA-1, Fura redTM AM and Fluo-4 NW.

121

EXPERIMENTAL

Reagents

Rosewell Park Memorial Institute (RPMI-1640) media was obtained from Hyclone Laboratories,

(Utah); sodium dodecyl sulfate (SDS) and analytical grade ammonium acetate were from Fisher

Scientific (NJ) and dimethyl sulfoxide (DMSO) anhydrous was from Sigma-Aldrich (St. Louis).

Phorbol myristate acetate (PMA) and ionomycin were obtained from Calbiochem (USA). All aqueous solutions were prepared in 18 Ω Milli Q water. The fluorescent dyes, FluoZinTM-3 AM,

Newport GreenTM DCF, Fura redTM AM, Calcium Green-1TM AM, and Oregon Green® 488

BAPTA-1 were purchased from Molecular Probes (Life Technologies, Grand Island, NY);

Zinpyr-1 was from Santa Cruz Biotechnology (Santa Cruz, CA) and Zinquin ethyl ester from

Enzo Life Sciences (Farmingdale, NY), Fluo-4 NW (No-Wash) Ca2+ assay kit was kindly provided by Dr. William Miller (University of Cincinnati Medical Center).

Cell line

Bone marrow macrophage cell line (BMC) was obtained from Dr. Kenneth Rock145 and cultured in tissue culture flasks with media (RPMI + 10% fetal bovine serum + gentamycin) at 37°C with

6 5% CO2. Confluent cultures were passaged every 3-4 days. 1.25 X 10 cells were plated in 2 ml media in 35 X 10 mm tissue culture dishes for 24 h prior to treatment of BMCs with metal binding dyes.

Treatment with fluorescent dyes

The Zn2+ and Ca2+ binding dyes were dissolved in anhydrous DMSO and stored in the dark at -

20°C. BMCs were washed twice with Ca2+ and magnesium free phosphate buffered saline (PBS) containing 2 mM EDTA to chelate extracellular metals. Cells were then treated with the indicated concentration of dyes (Table 1) or with DMSO control in RPMI media without serum

122 or in assay buffer (for Fluo-4 NW, as per manufacturer‟s instructions) and incubated for 30 min

2+ 2+ at 37°C with 5% CO2 in the dark. For analysis of Ca binding by the dyes under a Ca release stimulus, BMCs were treated with 50 ng/ml PMA, and 500 ng/ml ionomycin for 10 min to trigger intracellular Ca2+ release prior to addition of the dye. Post-incubation, BMCs were washed twice with Hank‟s balanced salt solution (HBSS) to remove unbound and extracellular dye and lysed with 0.1% SDS in Milli Q deionized water on ice for 20 min in the dark. Cell lysates were filtered through a 0.22 µm membrane and 50 µl of cell lysate was immediately injected into the HPLC-ICP-MS system for analysis.

Size exclusion chromatography

The separations were carried out with an Agilent 1100 HPLC (Santa Clara, CA, USA) equipped with a solvent degasser, binary pump, thermostated auto sampler, thermostated column compartment, diode array UV-Vis detector equipped with a semi-micro flow cell and a diode array fluorimetric detector equipped with a standard flow cell. The system was controlled using

Chemstation software.

The size exclusion column used was a TSK gel 3000SW 7.5 x 300 mm (Tosoh Bioscience,

Germany) (exclusion range >600kDa-10 kDa), the mobile phase was 50 mM ammonium acetate pH 7.4 at a flow rate of 0.5 ml min-1. The size exclusion column was calibrated using a UV detector (wavelength, 280 nm) using a gel filtration standard mixture Bio-Rad Laboratories (Life

Science Research, CA, USA, thyroglobulin, 670kDa; γ-globulin, 158 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa; and vitamin B12, 1.3 kDa) r = 0.997.

UV-Vis, FL and ICP-MS detection

Three detectors were used in tandem; first the UV-Vis detector generated spectra from 210 to

600 nm in 2 nm steps, at a speed of 2 spectra per second. As second detector a diode array

123 fluorescence detector (Santa Clara, CA, USA) was used and conditions were adjusted according to the optimum excitation/emission wavelengths recommended for each specific dye. Emission spectra were collected at a speed of one spectrum every five seconds. The details are shown in

Table 1.

The effluent from the HPLC was directly connected to the ICPMS nebulizer by a 0.17 mm ID

PEEK capillary. The specific detection of elements in the size exclusion chromatography (SEC) separated cell lysates was carried out in an Agilent 8800x ICP-MS (Agilent Technologies, Santa

Clara, CA, USA), equipped with a micro mist nebulizer, a cooled double pass spray chamber and standard torch. The operating conditions were as follows: forward power, 1550 W; plasma gas flow rate 15 L min-1, carrier gas flow, 0.91 L min-1; makeup gas 0.12 L min-1; collision gas, He at

4.0 mL min-1; quadrupole bias, -16.0 V; octopole bias -18.0 V for a +2 V energy discrimination setting. The isotopes monitored were 23Na, 27Al, 34S, 39Mg, 44Ca, 51V, 55Mn, 56Fe, 59Co, 60Ni,

63Cu, 66Zn and 114Cd.

Proteomics analysis

Protein identification experiments were carried out on the control samples, as a shot-gun bottoms-up approach. In brief, the different fractions of the SEC chromatogram were collected off-line for seven consecutive injections of 100 microliters each and the fractions were concentrated by freeze-drying (a fraction could be one or more than one SEC peak). The pellets obtained after freeze drying were re-suspended in 25 µL of 50 mM ammonium bicarbonate, then

2 µL of 100 mM dithiothreitol (DTT) were added as reducing buffer and the mixture was heated at 95 °C for 5 minutes. After cooling the sample, an alkylation step was carried out to protect the thiol groups of the cysteine residues by adding 3 µL of 100 mM iodoacetamide. The mixture was then kept in the dark at room temperature for 20 minutes. After alkylation, 2 µL of modified

124 sequence grade trypsin solution were added and incubated at 37 oC for 2 hours and then 2 µL of additional trypsin was added to complete the reaction, followed by incubation at 37 oC for 12 hours. 1.0 µL of formic acid was added to stop the reaction, and the solution was ultra-filtered through a 10 kDa filter to remove the undigested proteins and the unreacted trypsin. Filtrate was analyzed by nanoLC-ESI-ITMS.

An Agilent 6300 Series HPLC-CHIP-ESI-Ion Trap XCTsystem (Agilent Technologies,

Santa Clara, CA) coupled to an Agilent model 1200 LC, equipped with both capillary and nano pump, was used for peptide identification. Three microliters of sample was loaded via the capillary pump onto the on-chip enrichment column. The chip used consists of a Zorbax 300SB

C18 enrichment column (4 mm x 75 µm, 5 µm) and a Zorbax 300SB C18 analytical column (150 mm x 75 µm, 5 µm, Agilent Technologies, Santa Clara, CA, USA). Samples were loaded onto the enrichment column at a flow rate of 3 µL min-1 with a 97:3 ratio of solvent A (0.1% FA (v/v) in water) and B (90% ACN (acetonitrile), 0.1% FA (v/v) in water). After the enrichment column was loaded, the on-chip microfluidics switched to the analytical column at a flow rate of 0.3 µL min-1. The following gradient conditions were used in the analysis: 0–5 min, 10% B; 5–80 min,

40%B; 80–100 min, 70% B; 100–110 min, 0% B. Full scan mass spectra were acquired over the m/z range 150–2200 in the positive ion mode. For MS/MS experiments, experimental conditions consisted of: m/z range: 150–2200; isolation width: 2 m/z units, fragmentation energy: 30–200%, fragmentation time: 100 ms.

125

RESULTS

BMCs were used to trace the fluorescent behavior of dyes as a function of exchangeable metal or metal moiety. The fluorescence of dyes was evaluated primarily in two ways; the low molecular mass (LMM) response, and the target metal specificity by comparing size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) detection with

SEC-fluorescence (SEC-FL). Cells, rather than aqueous solutions, were chosen to provide an evaluation of metal-dye interactions and fluorescence responses in a cellular environment since aqueous solutions and cell matrices are markedly different.

The use of UV-Vis, fluorescence and ICP-MS as online tandem detectors provides information about the binding of dyes to specific molecular masses eluted from SEC column.

The UV-Vis absorbance spectra along with the SEC retention time can be used to distinguish proteins, nucleic acids, peptides and LMM compounds; while the ICP-MS provides a very specific and sensitive signal for the metal elution profiles, from the high molecular mass (HMM,

>600 kDa) to the LMM regions. The simultaneous appearance of ICP-MS and dye fluorescence responses in the LMM region is indicative of specific dye binding to a particular labile metal.

However, in the findings shown below, dye fluorescence was not always detected in the same molecular mass region where the metal was unambiguously identified by ICP-MS. This is an important observation, particularly in the LMM region where labile metals are expected to appear in the SEC chromatogram.

For dyes that recognize the labile fraction of a particular metal, the SEC-FL retention time should correspond to the LMM region of the SEC (lower than 5 kDa, at or after 22 min) and it is expected that fluorescence will be seen only in the LMM chromatographic regions and only if there is metal association with the dye. Any fluorescence signal before 22 min corresponds to

126 an association between the dye and the metal at HMM, in some cases, possibly proteins. Any

SEC-FL signal differing from the SEC-ICP-MS signal of the metal in question at a certain molecular mass range shows a lack of specificity for a particular metal or selectivity to one or more species of the same metal and may also represent background fluorescence of the dye.

All the experiments were carried out in biological triplicates on different days and produced similar results. The selection of BMCs was based on our previous experience with SEC-ICP-MS using this cell line for metallomics studies in the lab. The unstained cell lysates of BMCs served as a negative control and showed no FL signal at any molecular fraction (Supplementary

Figure 1). The ICP-MS and UV-Vis emission profiles show the distribution of Fe, Cu, Zn, Ca and Ni across high, medium and low molecular mass regions.

Zn2+ binding dyes

We selected the dyes that have been popularly used in imaging this metal to determine the relative response to Zn bound to larger biomolecules versus labile Zn2+. BMCs were treated with

Zn2+ dyes, followed by analysis of the SEC-FL response of the dye in correlation with the SEC-

ICP-MS signals for detection of Zn, Cu, Fe, Ni and S.

Detection of labile Zn2+

The goal of numerous Zn imaging studies has been to target the labile Zn2+ fraction to observe its behavior under varied stimuli. Though the total cellular zinc concentration is in the micromolar range, only picomolar quantities exist in labile form, making studies on this fraction challenging.

Therefore, we evaluated the propensity of Zn dyes to recognize the labile Zn2+ fraction in an intracellular environment.

127

Among the four dyes analyzed, Zinpyr-1 produced the highest fluorescence response corresponding to the labile Zn2+ fraction. Treatment of BMCs with Zinpyr-1 resulted in a strong

SEC-FL signal at 22 min (Figure 1a) that correlated with detection of a small, labile or LMM

Zn2+ fraction evident in the SEC-ICP-MS chromatogram (Figure 2a). An SEC-ICP-MS response for other metals was not detected in this region, indicating a specificity of the SEC-FL response of Zinpyr-1 for the detection of labile Zn2+. This signal represents the only anticipated response of the dye in terms of its usual applicability to determine labile Zn2+. Since the response of

Zinpyr-1 was relatively low in the HMM region, the dye exhibited highest selectivity to the

LMM fraction or to labile Zn2+.

FluoZinTM-3 AM is a widely used dye in imaging labile Zn2+ in cells13, 18, 25, 146. We therefore sought to determine the selectivity and specificity of this dye for the metal. The SEC-

FL response produced by FluoZinTM-3 AM at the ~22 min region representing labile Zn2+ was 11 fold weaker than the response generated upon interaction of the dye with HMM species in the

SEC-ICP-MS chromatogram (Figure 1b and 2b). In effect, the labile metal detection response of FluoZinTM-3 AM was ~10% of the overall SEC-FL signal generated by the dye as measured by area under the curve of the fluorescence response. Thus, in bioimaging applications, interpretation of the total fluorescence signal produced by this dye as a representation of the labile Zn fraction may lead to an over estimation of exchangeable Zn2+ in the sample.

Newport GreenTM DCF has been used physiologically to visualize free Zn2+ in mast cells131. In buffered solutions, the dye fluorescence is enhanced in the presence of zinc, but it also binds chromium, nickel, titanium and zirconium147 and has a high affinity for Cu and Fe148.

The fluorescence behavior of this dye showed a different trend when compared to Zinpyr-1 and

FluoZinTM-3 AM. Interestingly, Newport GreenTM DCF generated two fluorescence responses at

128 the LMM region past 21 min. Although, a very small labile Zn2+ fraction was detected by SEC-

ICP-MS at 22 min in the chromatogram, the fluorescence response of this dye correlated with the detection of S throughout the chromatogram. Of note, the dye fluoresced in the LMM region at

26 min, which did not only lack Zn2+, but was a region where none of the metals analyzed were detected. However, ICP-MS analysis identified two S peaks at the LMM region that aligned exactly with the SEC-FL response of Newport GreenTM DCF (Figures 1c and 2c). This S binding behavior was unique to Newport GreenTM DCF, and was not observed with other Zn2+ dyes (data not shown). Since the active chelating portion of the dye is cation-specific, the SEC-

FL signals may result from dye binding to S containing species associated to a cation, not included in the suite of metals studied. The SEC-ICP-MS analysis of Newport GreenTM DCF coupled with its fluorescence response suggests that detection of physiologically low concentrations of labile Zn may not be a characteristic of the dye in an intracellular environment.

Next, we studied the ability of Zinquin ethyl ester, an analog of 6-Methoxy-(8-p- toluenesulfonamido) quinoline (TSQ), to recognize and bind labile Zn2+. Compared to Zinpyr-1 that produced strong fluorescence and Newport GreenTM DCF, which led to a relatively weaker fluorescence response, Zinquin ethyl ester generated a very weak SEC-FL signal in the 22 min fraction (Figure 1d) comprising LMM Zn2+ (Figure 2d). Further, there was a minor overlap of the SEC-FL signal at 22 min with the Zn and Cu SEC-ICP-MS peaks, although the fluorescence intensity of Zinquin ethyl ester at this region was very low. Thus, the fluorescence signal contributed by this dye in the LMM region was less than 2% of the chromatographic peak area.

In the context of specificity of Zn2+ binding dyes in the LMM region, Zinpyr-1 and

FluoZinTM-3 AM generated specific responses to the LMM Zn2+ fraction; the fluorescence signal generated by Zinpyr-1 was stronger than that of FluoZinTM-3 AM in this region. On the other

129 hand, the association of Zinquin ethyl ester with the LMM region was minimal and generated an extremely weak response in the labile Zn2+ fraction. Newport GreenTM DCF demonstrated a lack of specificity to Zn2+ in the intracellular environment of BMCs, based on the association of the

SEC-FL signal with the detection of S in the chromatogram.

Binding behavior of Zn2+ dyes in high and mid molecular mass regions

In general, Zn2+ dyes poorly fluoresced in the LMM region, with the exception of Zinpyr-1.

Despite a weak signal, cellular microscopic imaging analysis of Zn using these dyes leads to a bright and detectable fluorescence signal13, 16, 131, 149. This prompted us to speculate that a majority of the fluorescence response may be generated upon association of the dye with higher molecular masses that elute prior to 22 min in the SEC-ICP-MS chromatogram.

Upon observing the fluorescence behavior of the Zn2+ dyes throughout the chromatogram, association of the dyes with higher molecular masses was evident. All the four dyes analyzed manifested a strong SEC-FL response in the HMM region (>600kD for the SEC column used) eluting at 9 min (Figures 1a-1d). However, there was an apparent difference in the fluorescence intensity generated by individual dyes in this region. In this regard, although

Zinpyr-1 produced an SEC-FL signal at 9 min, it was relatively weaker than Newport GreenTM

DCF. FluoZinTM-3 AM and Zinquin ethyl ester generated a strong fluorescence upon association to biomolecules in this region, indicating a greater affinity of these dyes for metals in the HMM fraction. The comparative intensity in fluorescence signals generated by the dyes in this region was Zinpyr-1 < Newport GreenTM DCF < FluoZinTM-3 AM < Zinquin ethyl ester from lowest to the highest response, indicating that Zinquin ethyl ester exhibited the strongest SEC-FL response in the HMM region. This fraction constitutes biomolecules such as large or multi-protein

130 complexes, nucleic acids, polysaccharides, membrane structures and incompletely lysed organelles that may be minimally retained based on the short elution time (void volume for this column indicating no retention). With regard to specificity of the Zn2+ dyes in the HMM fraction, all the metals analyzed including Zn, Fe, Cu and Ni were detected between 9-11 min of the SEC-

ICP-MS chromatogram. Although the SEC-FL response of the dyes appeared to correlate with the peak for Zn at 9 min, there was a fluorescence overlap with the peaks for Fe, Cu and Ni

(Figures 1a-1d). Thus, the net fluorescence signal generated by the dyes in the HMM region may result from the association of the dye with more than one metal.

The Zn2+ binding dyes also exhibited differential fluorescence behavior in the mid molecular mass region (500 kDa – 10 kDa). SEC-ICP-MS analysis showed peaks for Cu as well as Zn between 11 and 20 min of the SEC chromatogram (Figure 1a). Based on proteomic analysis, the Zn and Cu binding peak at 20 min contained the metal binding protein, metallothionein (Supplementary Table S1). Zinpyr-1 exhibited low fluorescence in the high and mid molecular mass regions (Figure 1a). The weak SEC-FL signals produced by FluoZinTM-

3 AM resulted from an association of the dye with a fraction of ~158 kDa at ~15 min and of ~20 kDa at 20 min, both of which correlated with the retention time of Zn and Cu (Figure 1b).

Newport GreenTM DCF fluoresced in the mid molecular mass region at 20 min correlating with the Zn, Cu and S peaks observed by SEC-ICP-MS (Figure 1c). The responsiveness of the dye to

S peaks throughout the chromatogram suggested that fluorescence was unlikely a result of specific Zn2+ binding. For Zinquin ethyl ester, multiple fluorescence signals were observed between the HMM and mid molecular mass fractions ranging from ~600 kDa to 100 kDa

(Figure 1d). The elution profile of the fluorescence signals corresponded to retention time for Zn

131 detected by SEC-ICP-MS, suggesting interaction of this dye with Zn-associated cell organelles, membrane structures or complex proteins in the HMM region.

Ca2+ binding dyes

Several dyes have been developed to analyze the flux of Ca2+ in single cells as well as in high throughput assays122, 133, 136, 150, 151. We found that under the „resting state‟ a majority or all of the intracellular Ca2+ appeared in the LMM fraction of the SEC-ICP-MS chromatogram as labile

Ca2+ (Supplementary Figure S1).

Detection of labile Ca2+

To determine the Ca2+ binding ability of the dyes at resting state, BMCs were treated with four different fluorescent probes, under homeostatic conditions without exogenous stimulation. The

SEC-ICP-MS response identified a distinct Ca2+ signal in the LMM region for the BMC lysates as shown in the SEC chromatogram (Figures 3-6) that was comparable to BMCs not exposed to the dyes (Supplementary Figure S1). Though the selected dyes, Calcium Green-1TM AM,

Oregon Green® 488 BAPTA-1, Fura redTM AM and Fluo-4 NW generated a robust SEC-FL response in BMCs, most of the dye fluorescence did not correspond to the labile Ca2+ signal unambiguously detected by SEC-ICP-MS.

Calcium Green-1TM AM generated a small fluorescence peak at 23 min that aligned with the SEC-ICP-MS detection of Cu2+ and Zn2+ in the LMM region (Figure 3a). Oregon Green®

488 BAPTA-1 produced very weak or negligible SEC-FL responses between 21 – 23 min that overlapped with low levels of Zn and Cu detected by SEC-ICP-MS (Figure 4a). These SEC-FL signals were a small contribution to the overall fluorescence generated by the dyes.

132

In BMCs treated with Fura redTM AM, the fluorescence generated in the LMM region was low. A sharp Ca2+ peak was identified in the SEC-ICP-MS trace at ~26.5 min. The only Ca2+ relevant fluorescence response of Fura redTM AM was the appearance of a small SEC-FL signal at ~26.5 min (Figure 5a). In contrast to Fura redTM AM, Fluo-4 NW exhibited a strong fluorescence in the LMM region between 22.5 to 25 min. This fluorescence signal aligned with a major peak for Zn, partially overlapping peak for Cu2+, but only a minor Ca2+ peak. A large Ca2+ peak eluted at the very LMM region. Of note, Fluo-4 NW generated an SEC-FL response, albeit small, in response to Ca2+ detection in this region (Figure 6a). Taken together, the SEC-FL contribution of Fura redTM AM and Fluo-4 NW upon non-specific binding was far greater than the fluorescence response produced by Ca2+ binding.

Binding behavior of Ca2+ dyes in high and mid molecular mass regions

To further probe the fluorescence response generated by the dyes, the SEC-FL signals detected by UV-Vis were analyzed throughout the SEC-ICP-MS chromatogram. As with the Zn2+ binding dyes, strong SEC-FL responses were detected with the four Ca2+ binding dyes in the HMM region (>600 kDa).

Calcium Green-1TM AM has been reported to exhibit a 14 fold increase in fluorescence upon exposure to Ca2+ along with a small shift in emission maximum152. A very strong SEC-FL signal was generated by the dye in the HMM region that corresponded to the detection of Zn by

SEC-ICP-MS; additionally the fluorescence signal overlapped partially with Cu and Fe in the void volume (Figure 3a). The response upon binding to molecules of high molecular mass constituted up to ~45% of the total fluorescence generated by the dye in BMC lysates as calculated by peak areas from the SEC-FL chromatogram. Two other SEC-FL peaks were

133 observed for Calcium Green-1TM AM in the mid and LMM regions at the retention times of 20 min and 23 min that did not constitute exchangeable Ca2+ indicated in blue in the SEC-ICP-MS trace. Instead these signals aligned with detection of Zn and Cu at both the retention times.

Calcium Green-1TM AM showed an atypical emission spectra with two emission bands in the

HMM region, but not in the mid molecular mass region, presented as a 3D projection of the fluorescence emission signals (Figure 3b). The UV-Vis emission isoplots offer an alternative way to view the emission behavior of the dye (Figure 3c). These data indicate that for imaging

Ca2+ using Calcium Green-1TM AM, acquisition of multi-wavelength data would be desirable.

In the HMM region, Oregon Green® 488 BAPTA-1 exhibited an SEC-FL response that aligned with SEC-ICP-MS signals for both Zn and Cu, and partially with that of Fe (Figure 4a).

Three additional SEC-FL signals were observed in the mid molecular mass regions, at ~12, 15 and 19 min, again, corresponding to the identification of Zn and Cu, but an Fe peak was not detected in this region. Of note, the intensity of SEC-FL response at 19 min containing two larger Zn peaks and a smaller Cu peak, identified as containing metallothionein (Supplementary

Table S1), was strong and comparable to the fluorescence response of the dye at 9 min in the

HMM region. The Ca signal detected by SEC-ICP-MS occurred almost entirely in the far LMM region, but none of the SEC-FL responses of the dye resulted from Ca2+ detection. These data indicate that the fluorescence generated by Oregon Green® 488 BAPTA-1 under homeostatic conditions may result from non-specific binding to Zn, Cu and Fe. Similar to Calcium Green-1TM

AM, the SEC-FL response of Oregon Green® 488 BAPTA-1 did manifest a change in emission spectral behavior upon metal recognition as observed in the 3D fluorescence projection and UV-

Vis emission isoplot (Figures 4b and 4c).

134

Fura RedTM responded with multiple SEC-FL signals in the HMM to mid molecular mass regions, however, the SEC-FL signal observed at the Ca2+ elution time shown by SEC-ICP-MS was negligible (Figure 5a). The SEC-FL emission in the HMM region at 10 min corresponded to

Zn and partially overlapped with that of Fe. Though the response of the dye was most intense at

~600 kDa, several lower intensity fluorescence signals were observed in the mid molecular mass regions between 11 min to 19 min where multiple Zn and Cu peaks were detected. This Ca2+ dye presented two distinct emission bands at 530 nm and 660 nm upon interacting with metals in the

HMM region, shown in the 3D projection and UV-Vis emission isoplot (Figures 5b and 5c) similar to those observed with Calcium Green-1TM AM.

Fluo-4 has an improved structural chemistry compared to Fluo-3, and the modification enhances fluorescence intensity upon Ca2+ binding152. This dye has been used extensively in studies ranging from imaging cell populations153 to high throughput drug screening assays for analysis of Ca2+ flux154. The fluorescence response of this dye was detected in the HMM region at ~10 min corresponding to chelation of non-Ca2+ containing species, for the most part, Zn and

Cu (Figure 6a). Fluo-4 NW also displayed a shift in the emission maxima, compared with the reported emission spectra, with a second band at 660 nm in the HMM region upon non-specific metal detection (Figure 6b and 6c). Collectively, under the physiological resting state of Ca2+ in

BMCs, the dyes exhibited a relatively high fluorescence intensity upon non-specific and non- selective binding to metals in the HMM region.

Fluorescence response of Ca2+ binding dyes under stimulation conditions

An application of Ca2+ imaging dyes is to follow Ca2+ changes as a result of enhanced influx or release from intracellular stores upon stimulation. Therefore, in Ca2+ imaging studies, the

135 fluorescence response of the dye prior to stimulation is corrected by setting the value to zero.

The selected Ca2+ binding dyes produced a series of mid molecular mass and HMM SEC-FL signals, unrelated to Ca2+ under homeostatic conditions. To investigate whether the lack of Ca2+ recognition was due to an inability of the dye to permeate intracellular organelles that store the metal, cells were stimulated with PMA and ionomycin to trigger Ca2+ release from organelles and to enhance influx155, 156. The Ca2+ related fluorescence response, post-stimulation, was compared with the resting control for two of the four dyes.

Exposure to PMA and ionomycin generated an increased influx of the metal, as is evident from the enhanced Ca2+ signal intensity observed in the SEC-ICPMS chromatogram (Figures 7 and 8). However, Fura RedTM staining of stimulated BMCs did not result in a corresponding substantial increase in the fluorescence signal in the region where Ca2+ was detected by ICP-MS

(Figures 7a-7c).

Next, we evaluated the behavior of Fluo-4 NW upon stimulation of BMCs to induce Ca2+ flux. PMA and ionomycin effectively enhanced the intensity of the SEC-ICP-MS response for

Ca2+ at the LMW region (Figure 8). For the specific case of Fluo-4 NW, a small enhancement in the SEC-FL response was seen upon stimulation, representing a 20% peak area increase at the region where Ca2+ was detected compared to the resting cells (Figures 8a-8c). This response was the only relevant signal generated by the dye upon binding Ca2+. However, stimulation of BMCs led to an elevated SEC-FL response by Fluo-4 NW at 9 min and 23 min in the HMM and LMM regions respectively, that did not correspond to Ca2+, but to Zn, Cu and Fe. The enhanced fluorescence of the dye in the presence of an exogenous stimulus is a confounding factor in confocal imaging, flow cytometry or high throughput analysis. The net increase in the fluorescence response above baseline, obtained upon stimulation, may not necessarily

136 correspond to specific alterations in Ca2+ flux triggered by the stimulus under study, but may result from non-specific interactions of the dye with other metals.

137

DISCUSSION

There is an increasing interest in the use of fluorescent probes for Zn and Ca analysis in biological systems. However, the metal interactions and binding behavior of these dyes in a complex cellular environment have been sparingly investigated. We selected commonly used metal imaging probes for Zn and Ca and obtained reproducible UV-Vis, FL and ICPMS signals by staining a BMC cell line using typical conditions employed for confocal imaging and flow cytometry. Lysis of the cells with 0.1% SDS on ice and immediate introduction to the HPLC with UV-Vis, SEC-FL, and ICP-MS detection was found to be an excellent way to track the fluorescent response of each dye, based not on the optical signal obtained as in imaging experiments, but on the association of the dyes with different metals and the varying molecular masses in the lysate fractions. With this approach using the three tandem detectors, it was possible to observe interaction of the dyes with metal-bound entities across a range of molecular masses. Most of the fluorescence responses did not result from labile metal detection in the

LMM region. A summary of the fluorescence behavior of the dyes is presented in Table 2.

The pool of exchangeable Zn2+ in cells appeared in the LMM region, however, the Zn2+ binding dyes, except Zinpyr-1, exhibited intense fluorescent signals in the HMM fractions. For the particular case of Newport GreenTM DCF, the two strongest fluorescent signals at LMM region were not related to Zn or any other metal analyzed. While Zinpyr-1 generated a response in the LMM region, FluoZinTM-3 AM was weakly responsive to labile metal and fluorescence from Zinquin ethyl ester largely resulted from recognition of metals in the HMM region. The chemical properties of small molecule Zn2+ probes may influence their propensity to fluoresce in the HMM region. Modification of the dye structure to facilitate entry into cells often results in the dye being trapped in vesicles or organelles after enzymatic cleavage by hydrolases157. One

138 such example is the Zinquin ethyl ester that complexes with Zn bound proteins and is retained intracellularly within vesicles158. The SEC-FL response of Zn2+ dyes in the HMM region may therefore represent dye-metal complexes retained into incompletely lysed intracellular vesicles that elute in the void volume of the SEC chromatogram. These data suggest that Zn2+ dyes not only recognize the labile Zn2+ fraction, but also respond effectively to Zn bound to higher molecular masses in an intracellular environment.

The four Ca2+ dyes tested exhibited very weak fluorescence in response to exchangeable

Ca2+ under homeostatic conditions. In fact a majority of the registered fluorescence responses from the Ca2+ dyes were in the mid molecular mass to HMM regions, and these were associated to Zn, Cu and Fe. It is possible to identify non-specific metal binding by the Ca2+ dyes in microscopic applications by observing the changes in the fluorescence emission spectra.

Intracellular Ca2+ ions may localize to organelles, such as the endoplasmic reticulum and mitochondria124, 134. This behavior may have resulted in non-specific and non-selective recognition of metal ions such as Zn, Cu, and Fe in the cytosol by the Ca2+ binding dyes. In response to a stimulus, Ca2+ flux is triggered and leads to enhanced import of the metal from extracellular spaces and release from organelles159, 160. Therefore, we analyzed response of dyes in the presence of a Ca2+ ionophore156, 161. While stimulation enhanced Ca2+ as detected by SEC-

ICP-MS in the LMM region, the SEC-FL produced by the dyes in this region remained the smallest signal, and did not increase in proportion to the additional Ca2+ released.

Specific detection of Ca2+ by fluorescent indicators is hampered by the presence of heavy metals, as they fluoresce strongly upon heavy metal binding162. It has been suggested that the use of the metal chelator NNNN-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) in cells to deplete heavy metal traces permits more accurate detection of Ca2+. TPEN however, chelates Zn

139 with strong affinity, and also binds Fe in cells163. Chelation of Zn and Fe to analyze Ca2+ flux in cells may lead to perturbations of numerous critical homeostatic cellular functions that depend on the availability of these metals20, 164. In this context, the fluorescence response of Ca2+ binding dyes has been tested in screening assays for drugs that modulate G-protein coupled receptors activity165 and to test drug resistant and sensitive cell lines166. The high SEC-FL response generated by the four Ca2+ dyes due to non-specific metal detection and non-selective binding to

HMM species, under resting as well as stimulated conditions indicates that the net fluorescence signal contribution in these assays may not exclusively result from alterations in the intracellular

Ca2+ pool.

We have tested the fluorescence response of Zn2+ and Ca2+ binding dyes using a bone marrow macrophage cell line. The relative metal composition, concentrations of each metal and their distribution may vary among other cell lines as well as primary cell types. These variations may lead to differential behavior of dyes depending on the cell type under examination, and a particular dye may be suited to imaging specific cell types. Nonetheless, a majority of the dyes have been widely applied in Zn2+ or Ca2+ analysis in phenotypically distinct cell populations such as immune cells13, 131, cardiomyocytes133, fibroblasts167, and neurons136, 168.

140

CONCLUSION

This study has addressed an important aspect in the use of small molecule fluorescent dyes for the detection of metals in a cellular environment. We analyzed the fluorescence behavior of commonly used Zn2+ and Ca2+ indicators to investigate their specificity for a particular metal ion and selectivity for one or more forms of a given metal in an intracellular environment. In the case of Zn2+ indicators, Zinpyr-1 fluoresced effectively in response to labile Zn2+, but FluoZinTM-3

AM, Newport GreenTM DCF and Zinquin ethyl ester were strongly responsive to metals bound to larger, complex biomolecules and weakly or negligibly responded to exchangeable Zn2+. In studies with Ca2+ indicators, the four dyes tested, Calcium Green-1TM AM and Oregon Green®

488 BAPTA-1, Fura redTM AM and Fluo-4 NW strongly fluoresced in response to larger bio- molecular complexes containing Zn, Cu and Fe. A majority of the cellular Ca2+ occurred in the labile form, but the Ca2+ binding dyes responded sub-optimally to this metal under both, resting and stimulated conditions. Collectively, the data obtained from analysis using a particular cell line indicate that fluorescence generated by chemical probes within cells may result from non- selective binding to labile and protein bound as well as membrane bound metals and/or from non-specific affinity for other divalent cations. These results emphasize that the use of metal binding dyes in bio-imaging applications should be supported by additional approaches to derive conclusions regarding the modulation of metals in a cellular environment.

141

ACKNOWLEDGEMENTS

The authors would like to thank Agilent Technologies for their continued support on chromatography and mass spectrometry. We thank Dr. W. Miller for providing us the Fluo-4

NW dye for Ca2+ studies and the NIH for support by grants # AI-094971, AI-106269 and a Merit

Review from Veterans Affairs.

AUTHOR CONTRIBUTIONS

K.S.V and J.L.F contributed equally, designed and performed experiments, analyzed and interpreted data and wrote the manuscript, K.S.V performed cell culture, staining and sample preparation for fluorescence analysis and proteomics experiment; J.L.F. performed chromatographic separations with UV-Vis, SEC-ICP-MS and ESI-IT-MS-MS for proteomics analysis and interpreted data; G.S.D. and J. C. designed and supervised the work. All authors contributed to manuscript preparation.

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

142

FIGURE LEGENDS

Figure 1: Specificity and selectivity of Zn2+ binding dyes

SEC-FL and SEC-ICP-MS chromatograms of (a) Zinpyr-1; (b) FluoZinTM-3 AM; (c) Newport

GreenTM DCF; (d) Zinquin ethyl ester; Fe, Ni, Cu, Zn and S signals are represented by solid lines and belong to the left Y axis; the SEC- FL signal is represented by the black line and corresponds to the right Y axis, fluorescence signal is offset to allow easy comparison; the MW markers are shown at the top of the chromatograms.

Figure 2: Zoom in of the LMM region of SEC-ICP-MS trace shown in Figure 1

Zoom in of the 15-30 min fraction of the SEC-ICPMS traces shown in Figure 1 for (a) Zinpyr-1;

(b) FluoZinTM-3 AM; (c) Newport GreenTM DCF; (d) Zinquin ethyl ester; three of the four dyes, except Newport GreenTM DCF, show a Zn2+ signal in the LMM region; the intensity of Zn2+ signal of LMM species is far less than that observed in the HMM regions.

Figure 3: Specificity and selectivity of Calcium Green-1TM AM

(a) SEC-FL and SEC-ICP-MS chromatograms of Calcium Green-1TM AM; Fe, Cu, Zn and Ca signals represented by solid lines belong to the left Y axis, while the SEC-FL signal represented by the black line corresponds to the right Y axis; the majority of the FL response is at 10 min

(HMM), and subsequent SEC-FL signals are apparent, but Ca2+ is not associated with any of these; (b ) 3D representation of emission wavelength, time and intensity; (c) UV-Vis emission isoplot of Calcium Green-1TM AM; signals at 10 min and 20 min are observed, the arrow indicates the second SEC-FL band at 660 nm in the HMM region.

143

Figure 4: Specificity and selectivity of Oregon Green® 488 BAPTA-1

(a) SEC-FL and SEC-ICP-MS chromatograms of Oregon Green® 488 BAPTA-1; Fe, Cu, Zn and Ca signals represented by solid lines belong to the left Y axis, while the SEC-FL signal represented by the black line corresponds to the right Y axis; SEC-FL signals are observed at

HMM and ~20 min, but Ca2+ is not associated with any of these; (b) 3D representation of emission wavelength, time and intensity; (c) UV-Vis emission isoplot of Oregon Green® 488

BAPTA-1; the SEC-FL signal shows a spectral behavior with two emission bands in the HMM region, the second band is indicated by the arrow.

Figure 5: Specificity and selectivity of Fura redTM AM

(a) SEC-FL and SEC-ICP-MS chromatograms of Fura redTM AM; Fe, Cu, Zn and Ca signals represented by solid lines belong to the left Y axis, while the SEC-FL signal represented by the black line corresponds to the right Y axis; the Ca2+ response is detected by ICP-MS at 26 min; the most intense SEC-FL signal is at HMM, with no Ca2+ response from ICP-MS; (b) 3D representation of emission wavelength, time and intensity; (c) UV-Vis emission isoplot of Fura redTM AM; the SEC-FL signal shows a change in spectral behavior at HMM with a second emission band, indicated by the arrow.

Figure 6: Specificity and selectivity of Fluo-4 NW

(a) SEC-FL and SEC-ICP-MS chromatograms of Fluo-4 NW; Fe, Cu, Zn and Ca signals represented by solid lines belong to the left Y axis, while the SEC-FL signal represented by the black line corresponds to the right Y axis; a small SEC-FL signal is associated with the detection of Ca2+ in the LMM region by ICP-MS; (b) 3D representation of emission wavelength, time and

144 intensity; (c) UV-Vis emission isoplot of Fluo-4 NW; the SEC-FL signal shows a second emission band at 10 min, indicated by the arrow.

Figure 7: Specificity and selectivity of Fura redTM AM with exogenous stimulation

(a) SEC-FL and SEC-ICP-MS chromatograms of Fura redTM AM in BMCs exposed to PMA and ionomycin stimulation; Fe, Cu, Zn and Ca signals represented by solid lines belong to the left Y axis, while the SEC-FL signal represented by the black line corresponds to the right Y axis; the

Ca2+ response detected by ICP-MS at 26 min shows an increase upon stimulation; a strong SEC-

FL signal is observed at HMM, with no Ca2+ response from ICP-MS; (b) 3D representation of emission wavelength, time and intensity; (c) UV-Vis emission isoplot of Fura redTM AM; the

SEC-FL signal shows a change in spectral behavior at HMM with a second emission band, indicated by the arrow.

Figure 8: Specificity and selectivity of Fluo-4 NW with exogenous stimulation

(a) SEC-FL and SEC-ICP-MS chromatograms of Fluo-4 NW in BMCs exposed to PMA and ionomycin stimulation; Fe, Cu, Zn and Ca signals represented by solid lines belong to the left Y axis, while the SEC-FL signal represented by the black line corresponds to the right Y axis; exogenous stimulation led to a small increase in SEC-FL signal associated with the detection of

Ca2+ in the LMM region by ICP-MS, but led to a larger increase in fluorescence in the HMM and mid molecular mass regions; (b) 3D representation of emission wavelength, time and intensity;

(c) UV-Vis emission isoplot of Fluo-4 NW; the dye exhibits a second emission band at 660 nm in the HMM region at 10 min, indicated by the arrow.

145

FIGURES

146

147

148

149

150

151

152

153

Table 1: FLD conditions for each probe studied, the excitation and emission were taken from the vendor instructions, while the spectra acquired were set 20 nm apart from the excitation wavelength to 750 nm.

Concen- Range for Excitation/Emission for tration Metal emission Name of the probe single wave length FL used Ion (2+) spectra , nm (µM) acquired , nm Zinpyr-1 5 Zn 490/530 510-750 FluoZinTM-3 AM 5 Zn 494/516 514-750 Newport GreenTM DCF 5 Zn 505/535 525-750 Zinquin ethyl ester 25 Zn 368/490 388-750 Calcium Green-1TM AM 5 Ca 506/530 526-750 Oregon Green® 488 5 Ca 466/494 486-750 BAPTA-1 Fura redTM AM 5 Ca 488/660 508-750 Fluo-4 NW 10 Ca 494/516 520-750

154

Table 2: Summary of selectivity results for the Zn2+ and Ca2+ binding probes. The signal at different molecular mass regions is represented by; + = 1-20%, ++ = 21-49%, +++ = >50% of total fluorescence signal.

% of the SEC-FL SEC-FL SEC-FL Name of total SEC- Target signal at signal at signal at Observations the probe FL signal HMM mid-MM LMM at LMM Zinpyr-1 Zn2+ + - +++ >70 FluoZinTM- Zn2+ +++ + + <10 3 AM SEC-FL Newport signals without GreenTM Zn2+ + - +++ >80 Zn signal at DCF LMW Zinquin Zn2+ +++ - + <2% ethyl ester No SEC-FL Calcium matched with Green-1TM Ca2+ +++ ++ + <5% ICP MS-Ca AM signal Oregon No SEC-FL Green® matched with Ca2+ ++ ++ + 10% 488 ICP MS-Ca BAPTA-1 signal

% of Ca-SEC- Fura redTM FL signal does Ca2+ +++ ++ + <5% AM not increase upon stimulation Fluo-4 Ca2+ ++ +++ + 15 % NW

155

Supplemental Information

SELECTIVITY AND SPECIFICITY OF SMALL MOLECULE FLUORESCENT

DYES/PROBES USED FOR THE DETECTION OF Zn2+ AND Ca2+ IN CELLS

&Julio A. Landero-Figueroa1, &Kavitha Subramanian Vignesh2, George Deepe3,4, and Joseph

Caruso1*

Supplemental Inventory

1. Supplementary Figure

Figure S1

2. Supplementary Table

156

Supplementary Figure S1: Unstained BMCs (negative) control

(a) SEC-FL and SEC-ICP-MS chromatograms of unstained BMC lysates; Fe, Cu, Zn, Ca and Ni represented by solid lines belong to the left Y axis, while the SEC-FL signal represented by the black line corresponds to the right Y axis; no SEC-FL signal is observed in the unstained control;

(b) UV-Vis emission isoplot of unstained BMCs.

157

Supplementary Table S1: Summary of protein IDs found in the 20 min SEC-ICP-MS fraction

Protein description MASCOT protein score

Actin, cytoplasmic 1 235

Pyruvate kinase isozymes M1/M2 207

Peroxiredoxin-1 OS=Mus musculus 179

78 kDa glucose-regulated protein 161 gamma actin-like protein [Mus musculus] 157 unnamed protein product [Mus musculus] 157

Plastin-2 OS=Mus musculus 149

Synaptic vesicle membrane protein VAT-1 147 histone H2B type 3-A [Mus musculus] 136

Annexin A1 OS=Mus musculus 128

Alpha-enolase OS=Mus musculus 123

Glyceraldehyde-3-phosphate dehydrogenase OS=Mus 120

Vimentin OS=Mus musculus 119 glyceraldehyde-3-phosphate dehydrogenase [Mus musculus] 116 peptidyl-prolyl cis-trans isomerase A [Mus musculus] 116

Ras-related protein Rab-7a OS=Mus musculus 113 unnamed protein product [Mus musculus] 112 heat shock protein 65 [Mus musculus] 110 alpha-fetoprotein [Mus musculus] 108

158 spermatid-specific [Mus musculus] 107 unnamed protein product [Mus musculus] 106

Beta-actin-like protein 2 OS=Mus musculus 105 malate dehydrogenase, mitochondrial precursor [Mus musculus] 103

Transgelin-2 OS=Mus musculus 102

Beta-enolase OS=Mus musculus 98 alpha-actin (aa 40-375) [Mus musculus] 96

ATP synthase beta-subunit [Mus musculus] 96

Atp5b protein [Mus musculus] 94

Moesin OS=Mus musculus 89 histone H2A [Mus musculus domesticus] 88 unnamed protein product [Mus musculus] 88

Heat shock cognate 71 kDa protein OS=Mus musculus 87

Metallothionein-2 [Mus musculus] 86

78 kDa glucose-regulated protein [Mus musculus] 84

Macrophage-capping protein OS=Mus 84 pyruvate kinase M [Mus musculus] 81 histone H1.2 [Mus musculus] 80

Heat shock protein HSP 90-beta OS=Mus musculus 2 79

Sulfated glycoprotein 1 OS=Mus musculus 78

Tubulin beta-3 chain OS=Mus musculus 74 profilin-1 [Mus musculus] 72

159 unnamed protein product [Mus musculus] 71

Myc basic motif homologue-1 [Mus musculus] 69 uncharacterized protein LOC433182 [Mus musculus] 69 unnamed protein product [Mus musculus] 69

40S ribosomal protein S5 [Mus musculus] 68 mCG15232, isoform CRA_b [Mus musculus] 68

Tubulin beta-5 chain OS=Mus musculus 67

Cathepsin B OS=Mus musculus 66

TI-225 [Mus musculus] 66 malate dehydrogenase, mitochondrial precursor [Mus musculus] 65

PREDICTED: ubiquitin-like protein ISG15-like [Mus musculus] 64

Tubulin beta-2A chain OS=Mus musculus 64 unnamed protein product [Mus musculus] 64

L-lactate dehydrogenase A chain isoform 1 [Mus musculus] 63

Annexin A4 OS=Mus musculus 60

Glyceraldehyde-3-phosphate dehydrogenase-like [Mus musculus] 60 unnamed protein product [Mus musculus] 59

ADP/ATP translocase 2 OS=Mus musculus 3 58 histone H2A.J [Mus musculus] 58

160

Annexin A5 OS=Mus musculus 55

Heat shock protein 75 kDa, mitochondrial OS=Mus musculus 55 unnamed protein product [Mus musculus] 55

Rho family GTPase [Mus musculus] 52 fetuin [Mus musculus] 51

Full=Galectin-3 51 unnamed protein product [Mus musculus] 51

65-kDa macrophage protein [Mus musculus] 50

RNA-binding protein FUS OS=Mus musculus 50 unnamed protein product [Mus musculus] 50 unnamed protein product [Mus musculus] 50 mCG1036674 [Mus musculus] 49

Stress-70 protein, mitochondrial OS=Mus musculus 49 tubulin beta-5 chain [Mus musculus] 49

Twinfilin-1 OS=Mus musculus 49 unnamed protein product [Mus musculus] 49 unnamed protein product [Mus musculus] 49

V-type proton ATPase subunit B, brain isoform

OS=Mus 49

ATP synthase subunit alpha, mitochondrial precursor

[Mus musculus] 48 endoplasmin [Mus musculus] 48

161 putative transmembrane glycoprotein [Mus musculus] 48

Transketolase OS=Mus musculus 48

Galectin-3 OS=Mus musculus 47 peroxiredoxin-4 precursor [Mus musculus] 47 unnamed protein product [Mus musculus] 47

ATP synthase subunit alpha, mitochondrial OS=Mus musculus 46

Macrophage scavenger receptor types I and II OS=Mus musculus 46

Fructose-bisphosphate aldolase A OS=Mus 45

Gamma-enolase OS=Mus musculus 44

Tropomyosin alpha-4 chain OS=Mus musculus 43

Aldehyde dehydrogenase, mitochondrial OS=Mus 42

Coiled-coil domain-containing protein 38 OS=Mus musculus 41

Cathepsin S OS=Mus musculus GN=Ctss PE=2 SV=1 39

40S ribosomal protein S8 OS=Mus musculus 38

Lysosome-associated membrane glycoprotein 1

OS=Mus 37

162

CHAPTER 5

Discussion and Future Directions

Review Article

ѱ Zinc Sequestration: Arming Phagocyte Defense Against Fungal Attack

Kavitha Subramanian Vignesh1,2†, Julio A. Landero Figueroa3†, Aleksey Porollo4, Joseph

A. Caruso3 and George S. Deepe, Jr2,5*

1Department of Molecular Genetics, Biochemistry, Microbiology and Immunology, University of Cincinnati, OH 45267 USA

2Division of Infectious Diseases, College of Medicine, University of Cincinnati, Cincinnati, OH 45267 USA

3University of Cincinnati / Agilent Technologies Metallomics Center of the Americas, Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221 USA

4Divisions of Rheumatology and Biomedical Informatics, Cincinnati Children‟s Hospital Medical Center, Cincinnati, OH 45229 USA

5Veterans Affairs Hospital, Cincinnati, OH 45220 USA

Contact: George S. Deepe, Jr. MD, email- [email protected], Phone-513-558-4706, Fax- 513-558-2089

Keywords: zinc, fungi, phagocytes, sequestration

ѱ Accepted, PLoS Pathogen Pearls, September 2013

163

Introduction

The innate immune system employs various defense mechanisms to combat invading microbes.

From a pathogen perspective, access to adequate nutrition is one of the fundamental requirements for survival within the host. The ability to counter microbial survival by restricting basic elements of growth, extending from amino acids to sugars and metals is referred to as nutritional immunity 118. The mechanisms of Zn2+ acquisition, transport and storage have been investigated in both prokaryotic and eukaryotic systems. In this review the total amount of zinc regardless of its chemical form will be referred to as Zn, and the labile fraction as Zn2+. From an immunological perspective, the primary focus has been on the impact of Zn2+ regulation on the numbers and function of lymphocytes and phagocytes and their correlation with susceptibility to infections, but, a dissection of the molecular details in these processes has been lacking. More recently, understanding the Zn2+ modulatory mechanisms and how they drive host-pathogen interactions at the molecular level has been a subject of intense scrutiny. This review will accentuate existing and novel insights into the roles of Zn in nutritional immunity and in phagocyte defenses against fungi.

Zinc takes center stage: A common requisite in host-pathogen interactions

Regulation of Zn homeostasis is essential for several host functions at multiple levels: i) for cellular processes including, but not limited to, transcription, translation, catalysis and cell division; ii) for countering Zn2+ deficiency or excess and, iii) for immunomodulatory responses in host-pathogen interactions. An estimated 10 percent or 2800 proteins in the human genome are Zn-dependent, implying a critical role for this metal in biological functions 169. In the immune system, Zn2+ regulation is of paramount importance as the development and function of innate and adaptive arms of immunity are influenced by this metal 9. Zn homeostasis established

164 by a balance in Zn2+ flux, intracellular distribution and storage, impacts phagocytosis, leukocyte recruitment, cytokine production, glycolysis and oxidation triggered in response to immune signals. Aberrant Zn2+ regulation in the circulation or in cells mitigates robust immune activation and leads to suboptimal host defenses. For example, Zn deficiency in humans with the genetic disorder acrodermatitis enteropathica is caused by Zn malabsorption and characterized by increased susceptibility to infections. An excess of Zn2+ diminishes T cell mitogenic responses 7.

Thus, an intact immune response requires strict Zn2+ regulation.

The fundamental requirement of Zn2+ for the function of several enzymes, transcription factors and structural proteins 170 is evident not only in mammals, but also in bacteria and fungi

171, in principal, due to the redox-inert property of this metal 128. Zn2+ enhances the synthesis of toxic secondary metabolites such as Aspergillus flavus mycotoxins that inhibit phagocytosis and cytotoxicity of T cells 172-174. Zn2+-dependent superoxide dismutases (SODs) produced by

Cryptococcus neoformans, Histoplasma capsulatum and Candida albicans are critical for scavenging superoxide radicals produced by phagocytes 175-177. These factors underscore the significance of Zn2+ acquisition for fungal pathogenesis and survival within the host. Thus, the struggle for Zn2+ between host and pathogen impacts survival of the invader and defense by the immune system.

Zinc acquisition strategies: Host versus Fungi

The immune system maintains Zn equilibrium via transporters, storage and binding (Figure 1).

While lower eukaryotes such as fungi possess fewer Zn2+ transporters 178, mammals have 24 transporters, called ZIPs (Slc39a, importers) and ZNTs (Slc30a, exporters). Some transporters manifest a ubiquitous expression pattern in several host cells, and others exhibit tissue specificity

165 and function irreplaceably in Zn2+ transport. For example, Slc30a1 is widely expressed in >12 organs, while Slc39a4 expression is restricted to the small intestine and kidney and is absolutely essential for dietary Zn absorption 179. Spatial organization of the transporters regulates Zn2+ in the cytosol and intracellular compartments including Golgi, mitochondria and zincosomes that are a source of exchangeable metal during deficiency 17. The remarkable complexity in Zn2+ transporters reflects the need for strict homeostasis and a regulatory system that responds to different biological stimuli in an organelle, cell and tissue specific manner. For example, interleukin-6 induces Zn2+ import via ZIP14 in hepatocytes 180, while granulocyte macrophage- colony stimulating factor (GM-CSF) triggers Zn2+ uptake via ZIP2 in macrophages181. The dependence of mammals on dietary sources for the metal implies the need for mechanisms that efficiently acquire Zn2+ and maintain regulated distribution in organ systems. Metallothioneins

(MTs) comprise a class of metal binding proteins that regulate Zn2+ and prevent intoxication.

MTs bind Zn2+ with picomolar affinity through 7 binding sites, one of which is more readily exchangeable, and interactions with glutathione, ATP or GTP mediates Zn2+ release 27. These properties facilitate a controlled exchange mechanism in infected phagocytes, where Zn2+ access to the microorganism needs to be restricted. Thus, phagocytes possess multiple mechanisms to manipulate Zn resources during infection.

To establish infection, fungi must adapt to limited nutrient availability upon encounter with the host. Upon phagocytosis, C. albicans triggers a transcriptional response signature reflecting a state of nutrient deprivation within macrophages 182. For pathogenic fungi, gaining entry into the host is associated with a transition from a Zn2+ sufficient external environment 183 to a lower Zn2+ containing milieu. Similarly, for opportunistic fungi such as C. albicans, the shift from a commensal to a pathogenic state may be accompanied by a dramatic paucity in Zn2+,

166 primarily due to Zn2+ restriction in the extracellular environment 184. Histoplasma capsulatum,

Cryptococcus neoformans and Blastomyces dermatitidis thrive in soil containing 30-350µM bioavailable Zn 155. Within macrophages, they are confronted with an environment containing only picomolar quantities of freely exchangeable Zn2+ 128. To thwart host defenses and establish infection, fungi must exert strategies to sense and respond to metal scarcity caused by sequestration into intracellular niches or binding to host proteins via high affinity interactions.

Mechanisms responding to Zn2+ availability in fungi include proteins that are directly affected by the presence or absence of Zn2+. For instance, Zn2+ inhibits DNA-binding activity of the Zn- responsive activator protein, Zap1p; however, a limiting milieu leads to transcription of Zap1p- dependent Zn2+ acquisition machinery. The upregulation of ZRT1 and ZRT2 transporters by

Zap1p under a Zn2+ deficient state is critical, as the absence of these importers diminishes fungal pathogenicity 178, 185. In Cryptococcus gattii, an ortholog of ZAP1 is upregulated by Zn2+ deprivation. Genetic deletion of ZAP1 impairs growth in a Zn2+-limiting environment, and mice infected with ZAP1 mutant yeasts exhibit increased survival 186. These findings emphasize a role for Zn2+ regulation in fungal virulence.

Although extracellular fungi do not directly compete for the pool of Zn2+ within cells, they must secure the metal from a restricted environment in infected tissue. A. fumigatus possesses ZrfA and ZrfB analogous to Zrt1p and Zrt2p that facilitate Zn2+ uptake in a low pH environment during deficiency 187. In a specialized mechanism described as the „zincophore system‟, C. albicans hyphae sequester host Zn2+ by secretion of pH regulated antigen-1, which re-associates with Zrt1p for subsequent import 185. Thus, multiple Zn2+ acquisition strategies in fungi collectively diminish vulnerability to host immunity. To establish virulence in vivo, these factors must contribute persistently to cope with metal scarcity induced by the immune system.

167

Host Zinc pool: Restricted access

Microbes are extremely sensitive to metal availability, and phagocytes have mastered mechanisms to curtail pathogen access to Zn2+. Despite our knowledge on Zn homeostasis, the manner in which the innate system modulates Zn2+ regulatory proteins in the context of fungal interactions and its influence on survival has been sparingly investigated.

In macrophages, a dual stimulus involving GM-CSF and H. capsulatum infection potently induces Zn2+ influx by ZIP2. The enhanced Zn2+ uptake may reflect at least two possibilities: i) a stress response during infection to support macrophage functions such as increased transcription and, ii) a mechanism to deprive extracellular yeasts of Zn2+ analogous to the induction of hypozincemia during bacterial sepsis. This phenomenon can be viewed as an opportunity for H. capsulatum to exploit Zn2+ elevation to capture more labile Zn2+ within the host. However, despite increased influx, GM-CSF creates a state of „deprived‟ intracellular free

Zn2+ by two mechanisms. First, GM-CSF causes Zn2+ localization in the Golgi, a shift associated with expression of exporters ZNT4 and ZNT7 that export cytosolic Zn2+ into the Golgi. Second, signaling via signal transducer and activator of transcription STAT3 and STAT5 triggers the production of MT1 and MT2 that constricts the labile Zn2+ pool by binding the metal181. The reduction in labile Zn caused by MT1 and MT2 is in contrast to the function of MT3 (Chapter 3), which facilitates binding and release of the metal, leading to increased availability. These studies highlight a fundamental Zn2+ sequestration property of transporters and MTs, which starves the pathogen and orchestrates a Zn2+ deprivation mechanism deployed by GM-CSF in macrophages

(Figure 2).

168

Spatial localization of Zn2+ transporters in the host potentially influences Zn2+ acquisition by intracellular fungi. ZNT4 enhances endosomal Zn2+ 23 that may be advantageous to the pathogen upon phagolysosomal fusion. In contrast, ZIP8 in T cells, deprives lysosomal Zn2+ by importing it into the cytosol 24. Though a role for importers in lysosomal Zn2+ deprivation has not been described in phagocytes, existence of such a mechanism would starve the pathogen of

Zn2+. Subcellular localization of transporters may be sensitive to the cellular microenvironment causing differential transport of Zn2+ in response to varying stimuli.

Apart from transporters and MTs, phagocytes produce calprotectin that binds Zn2+ with nanomolar affinity. Calprotectin in neutrophil extracellular traps (NETs) inhibits growth of C. albicans and also contributes to the fungistatic effect of plasmacytoid dendritic cells in A. fumigatus infection 33, 188. Conversely, an inability to regulate excess of Zn2+ jeopardizes pathogen survival. In exploiting this to the host‟s advantage, human macrophages impose Zn2+ intoxication in mycobacteria-laden phagosomes. Mycobacterial P1-type ATPases efflux the metal and alleviate Zn2+ poisoning 71. A role for heavy metal efflux pumps in fungal resistance to metal poisoning remains to be dissected. Thus, the immune system utilizes divergent Zn2+ restriction or intoxication mechanisms to combat infection. How immune cells preferentially utilize opposing stratagems against different microbial classes and the molecular cues that govern these decisions remain unclear. Collectively, Zn2+ modulation is a compelling arm of the innate system in restraining fungal persistence.

Zinc regulation: An impact beyond nutritional immunity

Regulation of Zn2+ shapes the functional attributes of innate defense, impacting phagocyte function beyond nutritional immunity. GM-CSF activated macrophages counter pathogen attack

169 by eliciting a dual defense strategy comprising Zn2+ restriction to H. capsulatum, while concurrently enhancing phagocyte effector function. Zn2+ abates superoxide production by

NADPH oxidase (Nox) by inhibiting hydrogen voltage-gated channel HV1181. Fungi scavenge superoxide radicals via Zn and Cu or Mn dependent SODs 175, 176. In activated macrophages,

MTs bind Zn2+ and create an environment deficient in Zn2+ ions, in effect, sustaining HV1 and

Nox function (Figure 2). In this milieu, H. capsulatum is susceptible to ROS181 , presumably due to an ineffectual Zn and Cu dependent SOD response. The extent of Zn2+ deprivation by MTs results in effective superoxide production, and may simultaneously compromise fungal SOD mediated defenses.

Collectively, Zn2+ restriction drives antifungal defense through a concurrent twofold effect, first, it induces Zn2+ starvation in the pathogen and second, it strengthens oxidative burst mediated defenses of the innate system. Thus, innate immunity is equipped with a variety of Zn2+ restriction strategies that function cooperatively to eliminate pathogens. As novel roles for metals are being described in dictating the outcome of immune regulation, we may be only beginning to appreciate the prominence of trace metals in immune defenses against infection.

170

Figure Legends:

Figure 1: Schematic of Zn regulation in phagocytes

Mechanisms of Zn regulation in phagocytes, grouped into three categories: Zn2+ transport, storage and binding: (A) Zn2+ transport across the cell membrane is mediated by ZIPs and ZNTs;

(B) Intracellular Zn2+ is transported into and stored in organelles such as endosomes, lysosomes,

Golgi and zincosomes by various transporters represented in the figure; the transporters that mediate Zn2+ flux across zincosomes have not been identified; (C) Zn2+ is bound and sequestered by intracellular or secreted metal binding proteins such as MTs and calprotectin.

Figure 2: Schematic of Zn regulation in activated macrophages infected with a fungal pathogen

Zn regulation in a GM-CSF activated macrophage leading to defense against fungal infection:

(A) GM-CSF binds to the GM-CSF receptor on infected macrophages, activates STAT3 and

STAT5 signaling and triggers transcriptional activation in the nucleus; (B) Induction of ZIP2 causes increased Zn2+ influx, which may support increased metabolic functions to cope with stress in the infected macrophage; (C) STAT3 and STAT5 induce expression of MTs that sequester labile intracellular Zn2+; (D) Zn2+ is mobilized into the Golgi apparatus, associated with increased expression of Golgi membrane transporters ZNT4 and ZNT7; (E) Speculated lysosomal Zn deprivation by influx into the cytosol by ZIPs; dotted arrow represents predicted sequestration of Zn2+ from this source by MTs; (F) Zn2+ inhibits proton flux via HV1, but the

„Zn2+ deprived‟ environment lifts the inhibitory action (shown on extreme right of the phagolysosomal membrane) and H+ generated by Nox activity is channeled into phagolysosomes effectively sustaining production of superoxide radicals by the enzyme; and, (G) The pathogen

171 senses a Zn2+ deprived environment and activates Zn-responsive transcription machinery to trigger Zn2+ import via fungal transporters and zincophore systems; ultimately, deficiency of

Zn2+ starves the pathogen of this metal and simultaneously enhances superoxide burst in phagocytes, culminating into inhibition of fungal growth.

172

Figure 1:

173

Figure 2:

174

FUTURE DIRECTIONS

We have dissected the mechanism by which Zn regulation by GM-CSF positively impacts macrophage defense against infection. The future directions in this work will focus towards three goals, first, to further understand the role of GM-CSF driven Zn modulation on T cell function, second, to thoroughly elucidate the pathway that leads to Zn regulation by IL-4 and third, to understand the broader impact of IL-4 driven Zn changes in macrophage immunity.

GM-CSF driven Zn modulation in T cell responses

Zn homeostasis may effectively regulate a balance in T cell populations and the effectiveness of

T cell responses. It has been suggested in vitro that chelation of Zn sustains STAT3 activation and supports differentiation of T cells into the Th17 lineage, simultaneously causing a reduction in the proportion of regulatory T cells. Since the adaptive response mounted during H. capsulatum infection is predominantly of the Th1 type, it is essential to investigate the impact of

Zn modulation by GM-CSF on the T cell responses.

Our studies highlight a role for GM-CSF in constricting the labile Zn fraction in macrophages. Not much is understood regarding regulation of Zn in various T cell populations.

First, the distribution of Zn proteome in T cells from H. capsulatum infected mice should be investigated to define the Zn-bound vs. labile Zn fraction in these cells. To determine the changes in Zn distribution in T cells during an immune response, a comparison to naïve T cells is essential. Further, the impact of GM-CSF on modulating Zn in T cells needs to be determined by neutralizing this cytokine and analyzing the T cells for Zn distribution. Since GM-CSF activates

STAT5 in T cells, we speculate that these cells will respond by sequestration of their labile Zn fraction. This effect is expected to be reversed upon neutralization of GM-CSF. Understanding

175 the consequences of Zn sequestration in T cells will yield important information about how these cells respond to alterations in Zn homeostasis, in terms of differentiation into specific lineages and activation. One may additionally speculate that GM-CSF neutralization skews the balance in

T cell populations. This may be a cause for altered Zn modulation or an effect of the same.

Secondly, the role of Zn modulation in phagocytes by GM-CSF may influence its interaction with T cells at various levels. These may include surface expression of MHC II,

CD80 and CD86 and other mediators that tune T cell activation and differentiation into distinct T cell lineages. We have studied Zn responses in macrophages, but the effect of this cytokine on

DCs is an area of interesting investigation. To probe this further, the Zn modulatory response in

DCs upon GM-CSF activation needs to be established as a primary step. Further, given the defined roles of DC-T cell interaction, one may speculate that Zn modulation in DCs and macrophages can effectively shape T cell responses during fungal infection.

Thus, the two potential areas of future investigations with GM-CSF include the direct regulatory role of GM-CSF on T cell responses and the indirect impact of Zn regulation in phagocytes on T cell differentiation and function.

Mechanisms of Zn regulation by IL-4

(i) Zn mobilization in IL-4 stimulated macrophages

Our experiments indicate that Zn is actively mobilized in macrophages under the influence of IL-

4. We propose to determine the source of Zn acquired by H. capsulatum, to investigate whether

IL-4 stimulation compromises labile Zn content in macrophages for preferential utilization by the

66 intracellular pathogen. Zn is an abundant isotope of Zn in biological systems. We have obtained purified forms of two different isotopes, 68Zn and 70Zn to address this question. By

176 differentiating bone marrow cells in media solely containing 68Zn, and H. capsulatum in media containing 70Zn as the Zn source, the total Zn content in differentiated macrophages and H. capsulatum can be directed to exist as 68Zn and 70Zn respectively. Mass based distinction by ICP-

189 68 MS efficiently differentiates between the three isotopes . In our in vitro model, Zn

66 macrophages will be cultured in media containing the regular isotope, Zn, followed by IL-4 stimulation and infection with 70Zn H. capsulatum. This distinctive system will answer several

66 important questions about Zn mobilization. First, appearance of Zn in macrophages during the

IL-4 priming stage or post infection will indicate active Zn influx into macrophages from the extracellular environment. Second, since MT3 is induced upon IL-4 stimulation and binds Zn,

66 68 the composition of Zn isotope ( Zn vs Zn) in the MT3 peak will reveal information about the source of Zn acquired by MT3. The peak may be largely composed of 68Zn, indicating utilization of cellular Zn resources by MT3 to acquire Zn. It is possible that new proteins synthesized

66 during IL-4 stimulation gain Zn from extracellular environment, which will appear as the Zn isotope in the chromatogram. Nonetheless, these analyses will uncover the source of MT3 bound

Zn. Analysis of the labile Zn signal at ~26 min will provide information about how Zn mobilization from the extracellular environment and intracellular sources influences the labile Zn resource upon IL-4 stimulation. Importantly, observations can be made by determining the Zn isotope distribution in yeasts recovered from IL-4 treated macrophages. If IL-4 creates a permissive environment for Zn acquisition by the yeast from macrophage sources, a diluting

70 68 66 effect of the Zn isotope with Zn and Zn from the macrophages will be observed. Moreover, this effect should be abolished in cells lacking MT3 and ZNT4 justifying their role as prime mediators in labile Zn increase and Zn acquisition by the intracellular pathogen. In the case of

177

GM-CSF activated macrophages, this technique may be employed to understand Zn deprivation from yeasts and sequestration by MTs.

(ii) Molecular mechanism of Zn release by IL-4

Our data indicate the involvement of MT3, ZNT4 and cathepsins as molecular intermediates in

IL-4 mediated Zn release. Although a role for MTs has been described in immune responses190, the proposition that transporters such as ZIP2 and ZNT4 possess immunological roles has not been investigated. Additional experiments described below will unearth the exact mechanisms by which the aforementioned intermediates bring about changes in Zn observed in IL-4 treated macrophages.

The small molecular weight of MTs (~7kD) and their ability to oligomerize has posed challenges in antibody mediated detection and proteomic analysis of the protein respectively. We will analyze Mt3-/- IL-4 treated macrophages compared to WT cells by SEC-ICP-MS. The absence of a Zn signal at 23 min in Mt3-/- macrophages will provide confirmatory evidence for retention of MT3 in that region. Further, the occurrence of labile Zn fraction at 26 min is expected to be diminished in Mt3-/- macrophages compared to WT, confirming the involvement of MT3 in Zn release. This study must however be additionally complemented with proteomic detection of MT3, because the absence of the protein may possibly alter Zn binding to other proteins in the chromatogram. We used ESI-MS-MS for studying the Zn proteome in macrophages to aid successful identification of MT2. However, in general, upon tryptic digestion the formation of disulfide bonds by peptides of MTs with other sulfur containing peptides poses a constraint in their detection. Therefore, we propose to use ESI-Q-TOF (ESI-quadrupole-time of

178 flight) for the detection of MT3 in macrophage lysates, which enables identification of proteins without the need for tryptic digestion.

Immunofluorescent detection of ZNT4 has been reported in literature23. In IL-4 treated macrophages, silencing ZNT4 leads to a decrease in labile Zn (Chapter 4). We speculate that IL-

4 causes localization of ZNT4 on the endosomal membrane, which may be instrumental in increasing labile Zn acquisition by H. capsulatum. The localization should be determined by immunofluorescent staining for ZNT4 as well as the early and late endosomal markers (early endosomal antigen 1 and mannose 6 phosphate receptor respectively) in IL-4 treated macrophages. In infected macrophages, ZNT4 co-localization may be observed with phagosomal markers Rab5 and Rab7. We observed an increase in the Zn signal at ~13-15 min in IL-4 treated macrophages. Preliminary proteomics analysis indicated the presence of transferrin receptor in this peak. The receptor imports Fe, but has also been reported to bind and transport Zn.

Transferrin receptor co-localizes with ZNT4 on endosomal membranes. We will test if co- localization of ZNT4 and transferrin receptor on the phagosomal membrane occurs in IL-4 treated macrophages. Although, colocalization will not identify Secondly, silencing ferritin will abate the increase in labile Zn despite the presence of ZNT4, suggesting the contribution of ferritin in increasing Zn availability either independently or by an interaction between the two proteins. Thus, investigating the role of ferritin will reveal important mediators in the Zn modulatory mechanism. It should be noted that silencing transferrin receptor may alter Fe levels in macrophages, in turn leading to altered Fe acquisition by the pathogen. In addition, analyzing the amassment of Zn by H. capsulatum in IL-4 treated macrophages lacking Slc30a4 expression alone and lacking both, Mt3 and Slc30a4 expression will dissect a role for these molecules in regulating yeast-associated Zn. We further speculate that silencing the expression of Mt3 and

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Slc30a4 may affect overall Zn homeostasis in macrophages. Specifically, the function of proteins that depend on MT3 and Slc30a4 for Zn acquisition, such as those in endosomes may be affected.

Cathepsins are a large family of proteases that degrade lysosomal proteins. Proteomic analysis of IL-4 treated H. capsulatum infected macrophages identified an abundance of cathepsin S peptide hits along with cathepsins B and D. We used a broad approach to target cathepsins and observed a decrease in labile Zn, a concominant increase in MT3-associated Zn signal, and attenuated Zn acquisition by H. capsulatum. The data suggest a role for cathepsins in

Zn release by MT3. However, the use of a specific approach, such as RNA interference will yield definite answers to the involvement of cathepsins. The most logical target would be cathepsin S, given its established role in macrophage lysosome protein degradation100 and identification in our proteomic analysis. The possibility of other members playing a compensatory role upon silencing cathepsin S will reveal interesting insights into alternative mechanisms of IL-4 mediated labile Zn release. In our studies on GM-CSF function, we have successfully silenced up to 3 genes simultaneously in a macrophage system. This approach is therefore realistic for combined silencing of CtsS, CtsB and CtsD genes. In addition, IL-4 treated macrophages must be tested for enhanced proteolytic activity compared to untreated controls to support a role for cathepsin activation. Fluorogenic substrates measuring cathepsin B, D and L activity exist, however these also respond to degradation by other proteases and a substrate to measure cathepsin S activity has not been developed.

(iii) Specificity of MT3 action in IL-4 driven labile Zn release

Our studies indicate a specific role for MT3 in Zn modulatory response by IL-4, however a role for MT1 and MT2 has not been ruled out. IL-4 stimulation increased Mt3 expression, but not that

180 of Mt1 and Mt2. Alterations in Zn binding to MT1 and MT2 peak at ~20 min in the SEC chromatogram were not observed. However, the lack of involvement of these members of the

MT family needs to be experimentally demonstrated by two approaches. First, we have obtained bone marrow macrophage cell lines from WT and Mt1-/-Mt2-/- mice, kindly generated by Dr.

Stuart Levitz (University of Massachusetts). Analyzing these cells for their Zn distribution behavior under the influence of IL-4 and H. capsulatum infection will reveal information regarding importance of MT3 in Zn release. Second, analyzing Zn distribution in Mt3-/- macrophages will provide a confirmatory evidence for the role of MT3, complement our silencing analysis, as well as enable elimination of a compensatory role for MT1 and MT2 in this system.

(iv) Alleviation of oxidative stress

In Chapter 2, we defined a role for Zn in inhibiting ROS production by NADPH oxidase. The generation of ROS in response to IL-4 in macrophages has been debated191, 192. Our premise suggests that the increase in labile Zn by IL-4 would impose an inhibitory effect on ROS production. The growth of several intracellular pathogens including H. capsulatum, is compromised by ROS. By making labile Zn available, MT3 may dually target inhibition of the

HV1 proton channel and enhance fungal growth by augmenting Zn availability. The effect of IL-

4 on ROS generation by WT and Mt3-/- infected macrophages, in comparison to no stimulus or

GM-CSF activation needs to be determined. It is expected that if IL-4 dampens ROS generation by driving Zn release from MT3, an aggravated superoxide response will be observed in Mt3-/- cells. It is also possible that oxidation of sulfur bonds in cysteine residues of MT3 promotes the release of Zn. This would imply that scavenging of ROS by MT3 leads to elevated labile Zn.

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Thus, an enhanced ROS response in Mt3-/- macrophages may result from lack of scavenging, rather than a lack of HV1 inhibition. Nonetheless, the combined outcome of MT3 action on ROS scavenging and/or HV1 inhibition may culminate in alleviated oxidative damage to the intracellular pathogen. As discussed before, the release of labile Zn may also facilitate optimal

SOD defenses by yeasts in IL-4 treated macrophages.

Zn regulation in alternative macrophage activation

Given the role for Zn in regulating several processes at the molecular level, availability and modulation of this metal can potentially impact fundamental properties of IL-4 signaling in macrophage differentiation and function.

(i) Labile Zn increase as marker for alternative activation

The activation of macrophages with GM-CSF induced deprivation of labile Zn, while exposure to TNF-α failed to manifest Zn regulation (Chapter 2). Stimulation with IL-4 gave rise to a distinct increase in labile Zn, and this phenomenon was not observed with „classically activating‟ macrophage cytokines. To broaden the scope of our observation, it is essential to test changes in

Zn distribution with other classical and alternatively activating cytokines such as IFN-γ and IL-

13 respectively. A shift from classical activation may be characterized by an increase in labile

Zn, indicating a transformation towards an alternative phenotype. It is essential to test the effect of IL-4 on Zn modulation in peritoneal and alveolar macrophages. Peritoneal macrophages possess a prominent labile Zn fraction under resting conditions. Whether this fraction of Zn exhibits an increase upon stimulation with IL-4 and the contribution of MT3 in this process should be determined. Elevation in labile Zn may have several advantages in supporting

182 permissive properties of M2 macrophages, which will be evident in the following sections. It must be noted that our studies on peritoneal macrophages showed enhanced Mt3 expression, but this was not specific to GM-CSF (Chapter 2). The labile Zn signal observed in resting peritoneal macrophages may be characteristic of the permissive properties of this population.

(ii) Zn regulation in lysosome function

The redox balancing role of Zn193 suggests that this metal controls critical functions in the lysosomal compartment. Exposure of macrophages to IL-4 results in decreased NO levels194, in part, due to an upregulation of Arg-1 that degrades the substrate arginine essential for NO synthesis. Another mechanism by which IL-4 may lower NO levels is via scavenging. MTs lower oxidative stress by binding to NO and releasing Zn167. We have observed that IL-4 induces

Mt3 which regulates the release of labile Zn. IL-4 driven MT3 regulation may serve as an additional mechanism of reducing NO in alternatively activated macrophages.

The redox properties of Zn bound to MTs may have similar effects in regulating superoxide radical production. The mechanism of Zn regulation by IL-4 is in sharp contrast to that of GM-CSF. The labile Zn release by IL-4 may be partly contributed by ROS quenching.

The levels of ROS in IL-4 treated macrophages may be dually influenced by the quenching property of MTs discussed above, as well as the release of labile Zn, which would inhibit HV1 driven Nox function. ROS levels in the absence of Mt3 in IL-4 treated macrophages may be higher as compared to Mt3+/+ macrophages indicating that the decrease in ROS is MT3 dependent. This would suggest the existence of a negative feedback loop in which elevated ROS triggers enhanced Zn release by MT3, which in turn shuts down superoxide production by inhibiting the HV1 channel.

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(iii) Inhibition of phagolysosomal fusion

The absence of MT3 has been associated with changes in markers of phagosome maturation.

MT3 may inhibit fusion of phagosomes to lysosomes by downregulating lysosome associated membrane protein (LAMP)1 and LAMP298. From the pathogen perspective, preventing fusion with lysosomes would result in a survival advantage, explaining the ability of IL-4 to favor fungal survival. However, whether IL-4 inhibits phagolysosomal fusion and the impact of Zn regulation in this process is unknown. We propose that IL-4 impedes phagolysosomal fusion by

MT3 driven Zn release. To test this, first, the impact of IL-4 on phagosomal maturation in infected macrophages must be determined by studying the co-localization of phagosome marker

Rab7 with lysosomal markers LAMP1 and LAMP2 in comparison to untreated macrophages or those stimulated with GM-CSF. If IL-4 imposes an inhibitory effect on phagosomal maturation, and the absence of MT3 would reverse it, such data will suggest a role for IL-4 driven Zn regulation in phagosomal maturation. Conversely, overexpression of Mt3 in macrophages may inhibit phagolysosomal fusion. Additionally, a direct role for Zn in thwarting phagosome maturation will support the premise that the observed changes result from Zn modulating properties of MT3, rather than the mere absence of this protein. In this case, the inhibitory effect of IL-4 on phagosomal maturation could be reversed by the Zn chelator TPEN, while an exogenous stimulation with Zn will ablate the process. These studies will reveal the impact of Zn in regulating phagosome maturation in alternatively activated- infected macrophages.

(iv) Giant cell formation:

Prolonged exposure of macrophages to IL-4 (~10 days) induces giant cell formation195. The role of multinucleated giant cells in dampening IL-4 driven host defenses remains elusive. In fungal

184 respiratory tract infection, giant cells surround yeast cells encapsulating them within granulomatous structures196.

Although the molecular events that drive giant cell formation by IL-4 have not been defined, the following evidence supports the idea that changes in Zn equilibrium can have an impact on the formation of multinucleated giant cells. The ability of IL-4 to trigger this effect in the absence of Mt3 should be examined by subjecting WT and Mt3-/- macrophages to prolonged

IL-4 exposure. The induction of giant cell formation can be microscopically examined and an aberration in this process will suggest a role for Zn regulation by IL-4. Several mediators have been identified as critical in giant cell formation. These include assembly of microtubules197, cell surface expression of β integrin1, β integrin2198 and E cadherin195. The established Zn dependent regulation of these molecules suggests that modulation of Zn by IL-4 may influence this process at multiple stages.

First, tubulin is a Zn-binding protein required for microtubular assembly, and the deficiency of Zn inhibits this process11. Thus, the acquisition of Zn and its release by MT3 may facilitate microtubular reorganization and fusion of macrophages in the presence of IL-4. Our data indicate that IL-4 triggers Mt3 expression immediately following exposure, but the kinetics of Mt3 expression need to be determined over time, because sustained expression of Mt3 may be required for IL-4 driven giant cell formation. Investigating microtubule assembly using tubulin specific antibodies in Mt3-/- macrophages compared to WT may reveal diminished assembly due to a lack of MT3 mediated Zn release. Second, the activation of β integrin by the urokinase receptor induced by IL-4 is dependent on Zn199. The effect of MT3 on the cell surface expression of β integrin should be investigated. If MT3 is involved in increasing labile Zn availability to promote β integrin surface expression, reduced levels of the protein will be observed in Mt3-/-

185 macrophages. This effect may also be reproduced by culturing WT macrophages in Zn depleted media, suggesting that the source of enhanced labile Zn availability by MT3 is via Zn import.

Lastly, the induction of E cadherin expression by IL-4 is absolutely essential in giant cell formation. IL-4 rapidly triggers Mt3 (Chapter 3) and induces E cadherin expression in a STAT6 dependent manner195. Overexpression of Mt3 in fibroblasts controls E cadherin expression200. It is possible that the early expression of E cadherin is STAT6 dependent, but prolonged induction of the protein by IL-4 may be dependent on Mt3. To test this, the dynamics of E cadherin occurrence on the cell surface of WT and Mt3-/- macrophages in the presence of IL-4 need to be determined. These studies can be additionally supported with Zn supplementation and Zn depletion in culture media to examine how Zn modulates the aforementioned molecular markers to shape the giant cell formation phenomenon. Conversely, analyzing the effect of restoration of

Mt3 in macrophages on microtubule assembly, cell surface expression of β integrins and E cadherin will establish the significance of Zn regulation by IL-4 in the formation of giant cells.

Thus, in addition to investigating the role of GM-CSF in Zn regulation, our future directions will uncover two major aspects of IL-4 function in phagocyte biology. First, the mechanism by which IL-4 drives Zn homeostasis and second, how Zn modulation by IL-4 globally impacts macrophage function. The data obtained from our first pursuit will identify clear roles for IL-4 in enhancing labile Zn, thereby weakening antifungal defense and the molecular mechanisms that drive this effect. Secondly, our proposition that IL-4 mediated changes in Zn homeostasis regulates fundamental properties of an alternatively activated macrophage, provides a unique perspective that relates metal modulation to phagocyte function.

Further, IL-4 has a prominent role in control of parasitic infection, wound healing and allergic responses. Knowledge of IL-4 driven Zn modulatory mechanisms in macrophages will provide a

186 platform for investigating the potential role of Zn in the broader impact of IL-4 function. In summary, probing the mechanisms by which IL-4 signaling shapes Zn modulation will provide novel insights into the fundamental aspects of innate responses produced by this cytokine.

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